Therapeutic inhibitor of vascular smooth muscle cells

ABSTRACT

Methods are provided for inhibiting stenosis or restenosis following vascular trauma in a mammalian host, comprising administering to the host a therapeutically effective dosage of a cytostatic agent and/or cytoskeletal inhibitor so as to biologically stent the traumatized vessel. Also provided is a method to inhibit or reduce vascular remodeling following vascular trauma, comprising administering an effective amount of a cytoskeletal inhibitor. Further provided are pharmaceutical compositions and kits comprising the therapeutic agents of the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/024,885, filed Dec. 18, 2001, which is a continuation off U.S.application Ser. No. 09/361,194, filed Jul. 26, 1999 now U.S. Pat. No.6,358,989, which is a divisional application of U.S. application Ser.No. 08/829,685 filed Mar. 31, 1997, now U.S. Pat. No. 5,981,568, thedisclosures of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Percutaneous transluminal coronary angioplasty (PTCA) is widely used asthe primary treatment modality in many patients with coronary arterydisease. PTCA can relieve myocardial ischemia in patients with coronaryartery disease by reducing lumen obstruction and improving coronaryflow. The use of this surgical procedure has grown rapidly, with 39,000procedures performed in 1983, nearly 150,000 in 1987, 200,000 in 1988,250,000 in 1989, and over 500,000 PTCAs per year are estimated by 1994(Popma et al., Amer. J. Med., 88: 16N-24N (1990); Fanelli et al, Amer.Heart Jour., 119: 357-368 (1990); Johnson et al., Circulation, 78(Suppl. II) II-82 (1988)). Stenosis following PTCA remains a significantproblem, with from 25% to 35% of the patients developing restenosiswithin 1 to 3 months. Restenosis results in significant morbidity andmortality and frequently necessitates further interventions such asrepeat angioplasty or coronary bypass surgery. As of 1993, no surgicalintervention or post-surgical treatment has proven effective inpreventing restenosis.

The processes responsible for stenosis after PTCA are not completelyunderstood but may result from a complex interplay among severaldifferent biologic agents and pathways. Viewed in histological sections,restenotic lesions may have an overgrowth of smooth muscle cells in theintimal layers of the vessel (Johnson et al., Circulation, 78 (Suppl.II): II-82 (1988)). Several possible mechanisms for smooth muscle cellproliferation after PTCA have been suggested Popma et al., Amer. J.Med., 88: 16N-24N (1990); Fanelli et al, Amer. Heart Jour., 119: 357-368(1990); Liu et al., Circulation, 79: 1374-1387 (1989); Clowes et al.,Circ. Res., 56: 139-145 (1985)).

Compounds that reportedly suppress smooth muscle proliferation in vitro(Liu et al., Circulation, 79: 1374-1387 (1989); Goldman et al.,Atherosclerosis, 65: 215-225 (1987); Wolinsky et al., JACC, 15 (2):475-481 (1990)) may have undesirable pharmacological side effects whenused in vivo. Heparin is an example of one such compound, whichreportedly inhibits smooth muscle cell proliferation in vitro but whenused in vivo has the potential adverse side effect of inhibitingcoagulation. Heparin peptides, while having reduced anti-coagulantactivity, have the undesirable pharmacological property of having ashort pharmacological half-life. Attempts have been made to solve suchproblems by using a double balloon catheter, i.e., for regional deliveryof the therapeutic agent at the angioplasty site (e.g., Nabel et al.,Science, 244: 1342-1344 (1989); U.S. Pat. No. 4,824,436), and by usingbiodegradable materials impregnated with a drug, i.e., to compensate forproblems of short half-life (e.g., Middlebrook et al., Biochem. Pharm.,38 (18): 3101-3110 (1989); U.S. Pat. No. 4,929,602).

At least five considerations would, on their face, appear to precludeuse of inhibitory drugs to prevent stenosis resulting from overgrowth ofsmooth muscle cells. First, inhibitory agents may have systemic toxicitythat could create an unacceptable level of risk for patients withcardiovascular disease. Second, inhibitory agents might interfere withvascular wound healing following surgery and that could either delayhealing or weaken the structure or elasticity of the newly healed vesselwall. Third, inhibitory agents which kill smooth muscle cells coulddamage surrounding endothelium and/or other medial smooth muscle cells.Dead and dying cells also release mitogenic agents that might stimulateadditional smooth muscle cell proliferation and exacerbate stenosis.Fourth, delivery of therapeutically effective levels of an inhibitoryagent may be problematic from several standpoints: namely, a) deliveryof a large number of molecules into the intercellular spaces betweensmooth muscle cells may be necessary, i.e., to establish favorableconditions for allowing a therapeutically effective dose of molecules tocross the cell membrane; b) directing an inhibitory drug into the properintracellular compartment, i.e., where its action is exerted, may bedifficult to control; and, c) optimizing the association of theinhibitory drug with its intracellular target, e.g, a ribosome, whileminimizing intercellular redistribution of the drug, e.g. to neighboringcells, may be difficult. Fifth, because smooth muscle cell proliferationtakes place over several weeks it would appear a priori that theinhibitory drugs should also be administered over several weeks, perhapscontinuously, to produce a beneficial effect.

As is apparent from the foregoing, many problems remain to be solved inthe use of inhibitory drugs to effectively treat smooth muscle cellproliferation. Thus, there is a need for a method to inhibit or reducestenosis due to proliferation of vascular smooth muscle cells followingtraumatic injury to vessels such as occurs during vascular surgery.There is also a need to deliver compounds to vascular smooth musclecells which exert inhibitory effects over extended periods of time.

SUMMARY OF THE INVENTION

The present invention provides a therapeutic method comprising theadministration of at least one therapeutic agent to a procedurallytraumatized, e.g., by an angioplasty procedure, mammalian vessel.Preferably, the therapeutic agent is a cytoskeletal inhibitor. Preferredcytoskeletal inhibitors in the practice of the present invention,include, for example, taxol and analogs or derivatives thereof such astaxotere, or a cytochalasin, such as cytochalasin B, cytochalasin C,cytochalasin A, cytochalasin D, or analogs or derivatives thereof. Theadministration of a therapeutic agent of the invention is effective tobiologically stent the vessel, inhibit or reduce vascular remodeling ofthe vessel, inhibit or reduce vascular smooth muscle cell proliferation,or any combination thereof. The administration of the therapeutic agentpreferably is carried out during the procedure which traumatizes thevessel, e.g., during the angioplasty or other vascular surgicalprocedure. The invention also provides therapeutic compositions anddosage forms adapted for use in the present method, as well as kitscontaining them.

Thus, one embodiment of the invention provides a method for biologicallystenting a traumatized mammalian blood vessel. The method comprisesadministering to the blood vessel an amount of a cytoskeletal inhibitorin a liquid vehicle effective to biologically stent the vessel. As usedherein, “biological stenting” means the fixation of the vascular lumenin a dilated state near its maximal systolic diameter, e.g., thediameter achieved following balloon dilation and maintained by systolicpressure. The method comprises the administration of an effective amountof a cytoskeletal inhibitor to the blood vessel. Preferably, thecytoskeletal inhibitor is dispersed in a pharmaceutically acceptableliquid carrier, e.g., about 0.1 to about 10 μg for cytochalasin B/ml ofaqueous vehicle, and preferably administered locally via a catheter.Another preferred embodiment of the invention is a cytochalasin oranalog thereof dispersed in a pharmaceutically acceptable liquid carrierat about 0.001 to about 25 μg per ml of aqueous vehicle. Preferably, aportion of the amount administered penetrates to at least about 6 to 9cell layers of the inner tunica media of the vessel and so is effectiveto biologically stent the vessel.

Preferred catheter administration conditions include employing acatheter to deliver about 4 to about 25 ml of a composition comprisingthe cytoskeletal inhibitor dispersed or dissolved in a pharmaceuticallyacceptable liquid vehicle. The cytoskeletal inhibitor is delivered at ahub pressure of about 3 to about 8 atm, more preferably about 4 to about5 atm, for about 0.5 to about 5 minutes, more preferably for about 0.7to about 3 minutes. Preferred hydrostatic head pressures for catheteradministration include about 0.3 to about 1.0 atm, more preferably about0.5 to about 0.75 atm. The amount of therapeutic agent is controlled soas to allow vascular smooth muscle cells to continue to synthesizeprotein, which is required to repair minor cell trauma, and to secreteinterstitial matrix, thereby facilitating the fixation of the vascularlumen preferably in a dilated state near its maximal systolic diameter,i.e., to provide a biological stent of the vessel. Preferably, thetherapeutic agent is administered directly or substantially directly tothe traumatized area of the vascular smooth muscle tissue.

The invention further provides a method for inhibiting or reducingvascular remodeling of a traumatized mammalian blood vessel, byadministering an effective amount of a cytoskeletal inhibitor to thetraumatized blood vessel.

As described hereinbelow, a dose response study showed that cytochalasinB had a two logarithmic therapeutic index (TI). A large therapeuticindex allows the diffusion of therapeutic levels of the agent from thedelivery system, e.g., an implantable device, without toxicity to cellsimmediately adjacent to the exit port of the system. Moreover, even atthe maximum concentration of cytochalasin B in a liquid vehicle, therewas little or no toxicity observed in cells adjacent to the deliverysystem. It was also found that cytochalasin B and taxol both inhibitintimal proliferation in vessels subjected to a procedural vasculartrauma. This inhibition results in a more rapid and completeendothelialization of the vessel wall following the trauma.

The invention also provides a therapeutic method comprising inhibitingdiminution of vessel lumen diameter by administering to a traumatizedvessel of a mammal an effective amount of a cytoskeletal inhibitor. Thecytoskeletal inhibitor is administered via an implantable device,wherein the implantable device is not a catheter which has a first and asecond expansile member, i.e., balloons, which are disposed on oppositesides of the vessel area to be treated in order to isolate the portionof the vessel to be treated prior to cytoskeletal inhibitoradministration. Preferably, the isolated portion of the vessel is notwashed to remove blood prior to cytoskeletal inhibitor administration(“bloodless angioplasty”). “Isolated,” as used above, does not meanocclusive contact of the actual treatment area by the catheter balloon,which is preferred in the practice of the present invention. Moreover,bloodless angioplasty, such as that described in Slepian, U.S. Pat. No.5,328,471, i.e., in which the region to be treated is washed, mayintroduce trauma or further trauma to the vessel, may increase thepotential for complications and is not necessary to achieve a beneficialeffect.

Thus, the invention further provides a method for inhibiting or reducingdiminution in vessel lumen volume in a traumatized mammalian bloodvessel. The method comprises administering to the blood vessel of amammal an effective amount of cytoskeletal inhibitor, wherein thecytoskeletal inhibitor is in substantially crystalline form and whereinthe crystals are of a size which results in sustained release of thecytoskeletal inhibitor. Preferably, the crystals are of a size of about0.1 micron to about 10 mm, preferably about 1 micron to about 25 micron,in size. Methods to determine the size of crystals useful for sustainedrelease are well known to the art. Preferably, the cytoskeletalinhibitor is administered in situ, by means of an implantable device,wherein the cytoskeletal inhibitor is releasably embedded in, coated on,or embedded in and coated on, the implantable device. Preferably, thecrystalline cytoskeletal inhibitor is releasably embedded in, ordispersed in, a adventitial wrap, e.g., a silicone membrane. Forexample, a preferred therapeutic implantable device of the inventioncomprises about 5 to about 70, preferably about 7 to about 50, and morepreferably about 10 to about 30, weight percent of a cytochalasin, e.g.,cytochalasin B or an analog thereof, per weight percent of theadventitial wrap. Another preferred therapeutic implantable device ofthe invention comprises about 1 to about 70, preferably about 2 to about50, and more preferably about 3 to about 10, weight percent of taxol oran analog thereof per weight percent of the adventitial wrap.Alternatively, a preferred therapeutic implantable device of theinvention comprises about 35 to about 70, preferably about 35 to about60, and more preferably about 35 to about 50, weight percent of taxol oran analog thereof per weight percent of the adventitial wrap.

Alternatively, the crystalline cytoskeletal inhibitor may be suspendedin a vehicle which yields a solution comprising the crystals, i.e., itis a saturated solution.

The invention further provides a therapeutic method. The methodcomprises administering to a traumatized mammalian blood vessel asustained release dosage form comprising microparticles or nanoparticlescomprising a cytoskeletal inhibitor, e.g., cytochalasin, taxol, oranalogs thereof. The sustained release dosage form comprising acytochalasin or analog thereof is preferably administered via animplantable device which is not a catheter used to perform bloodlessangioplasty. The amount administered is effective inhibit or reducediminution in vessel lumen area of the mammalian blood vessel. Thesustained release dosage form preferably comprises microparticles of 4to about 50 microns in diameter. The sustained release dosage form canalso preferably comprise about 2 to about 50, and more preferablygreater than 3 and less than 10, microns in diameter. For nanoparticles,preferred sizes include about 10 to about 5000, more preferably about 20to about 500, and more preferably about 50 to about 200, nanometers.

Also provided is a method comprising administering to a mammalian bloodvessel a dosage form of a cytochalasin or an analog thereof in anon-liquid vehicle or matrix effective inhibit or reduce diminution invessel lumen area of the mammalian blood vessel. Preferably the dosageform is a substantially solid dosage form. As used herein, “solid form”does not include microparticles, nanoparticles, and the like. Thenon-liquid vehicle or matrix preferably includes, but is not limited to,a gel, paste, or a membrane which comprises the cytochalasin or analogthereof.

Also provided is a kit comprising, preferably separately packaged, atleast one implantable device adapted for the in situ delivery,preferably local delivery, of at least one cytoskeletal inhibitor to asite in the lumen of a traumatized mammalian vessel and at least oneunit dosage form of the cytoskeletal inhibitor in a liquid vehicleadapted for delivery by said device. The administration of at least aportion of the unit dosage form to the traumatized vessel is effectiveto biologically stent the vessel, inhibit or reduce the vascularremodeling of the vessel, inhibit or reduce vascular smooth muscle cellproliferation, or any combination thereof.

Further provided is a kit comprising, preferably separately packaged, animplantable device adapted for the delivery of at least one therapeuticagent to a site in the lumen of a traumatized mammalian vessel and aunit dosage form comprising at least one cytoskeletal inhibitor, whereinthe administration of at least a portion of the unit dosage form iseffective to inhibit or reduce diminution in vessel lumen diameter ofthe vessel. The device is not a catheter which has a first and a secondexpansile member which are disposed on opposite sides of the region tobe treated so as to isolate a portion of the vessel to be treated priorto administration or wherein the isolated portion of the vessel is notwashed to remove blood prior to administration.

The invention also provides a kit comprising, separately packaged, animplantable device adapted for the delivery of at least one therapeuticagent to a site in the lumen of a traumatized mammalian vessel and aunit dosage form comprising an amount of microparticles or nanoparticlescomprising taxol or an analog thereof. Preferably, the kit alsocomprises a second unit dosage form comprising a pharmaceuticallyacceptable liquid carrier vehicle for dispersing said microparticles orsaid nanoparticles prior to delivery. The delivery of the dispersedmicroparticles or nanoparticles to the traumatized mammalian vessel iseffective to inhibit or reduce diminution in vessel lumen diameter inthe vessel.

Yet another embodiment of the invention is a pharmaceutical compositionsuitable for administration by means of an implantable device. Thecomposition comprises an amount of a cytochalasin or analog thereofeffective to inhibit or reduce stenosis or restenosis of a mammalianvessel traumatized by a surgical procedure and a pharmaceuticallyacceptable non-liquid release matrix for said cytochalasin. Preferably,the release matrix comprises a gel, paste or membrane.

Also provided is a unit dosage form. The unit dosage form comprises avial comprising about 10 to about 30 ml of about 0.001 μg to about 25 μgof a cytoskeletal inhibitor, preferably a cytochalasin, per ml of liquidvehicle, wherein the unit dosage form is adapted for delivery via animplantable device, and wherein the vial is labeled for use in treatingor inhibiting stenosis or restenosis. Preferably, the unit dosage formcomprises a vial comprising about 10 to about 30 ml of about 0.01 μg toabout 10 μg of cytochalasin B per ml of liquid vehicle. Thus, the volumepresent in a vial may be greater than, or about the same as, the volumepresent in the implantable device. Likewise, the volume present in theimplantable device may be greater than, or about the same as, the volumeadministered. Similarly, the volume administered may be greater than, orabout the same as, the volume which has a beneficial effect.

Further provided is a unit dosage comprising a vial comprising acytostatic amount of a cytoskeletal inhibitor in a pharmaceuticallyacceptable liquid vehicle. Preferably, the cytoskeletal inhibitorcomprises a cytochalasin, taxol, or an analog thereof.

The invention also provides therapeutic devices. One embodiment of theinvention comprises a therapeutic shunt comprising an amount of acytoskeletal inhibitor effective to inhibit stenosis or reducerestenosis following placement of the therapeutic shunt. Anotherembodiment of the invention comprises therapeutic artificial graftcomprising an amount of a cytochalasin or analog thereof to inhibitstenosis or reduce restenosis following placement of the graft. Yetanother embodiment of the invention comprises a therapeutic adventitialwrap comprising an amount of a cytoskeletal inhibitor effective toinhibit stenosis or reduce restenosis following placement of the wrap.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph of vascular smooth muscle cells of a24-year-old male patient.

FIG. 1B is a photomicrograph of vascular smooth muscle cells in anartery of a 24-year-old male patient with vascular smooth muscle bindingprotein bound to the cell surface and membrane. The patient received thevascular smooth muscle binding protein by i.v. administration 4 daysbefore the arterial tissue was prepared for histology.

FIG. 2 depicts a first scheme for chemical coupling of a therapeuticagent to a vascular smooth muscle binding protein.

FIG. 3 depicts a second scheme for chemical coupling of a therapeuticagent to a vascular smooth muscle binding protein.

FIG. 4A graphically depicts experimental data showing rapid binding ofvascular smooth muscle binding protein to marker-positive test cells invitro.

FIG. 4B graphically depicts experimental data showing rapid binding ofvascular smooth muscle binding protein to vascular smooth muscle cellsin vitro.

FIG. 5A presents graphically experimental data showing undesirablecytotoxicity of even low levels of therapeutic conjugate (i.e.,RA-NR-AN-01), and the free RA therapeutic agent, when vascular smoothmuscle cells were treated for 24 hours in vitro.

FIG. 5B graphically presents experimental data showing the effects ofRA-NR-AN-01 therapeutic conjugate on metabolic activity ofmarker-positive and -negative cells. The data show undesirablenonspecific cytotoxicity of the conjugate for all these cells in a 24hour treatment in vitro. The non-specificity results from extracellularhydrolysis of the coupling ligand which exposes the tested cells to freedrug.

FIG. 6A graphically depicts experimental data showing undesirablenonspecific cytotoxicity of PE-NR-AN-01 therapeutic conjugate formarker-positive and marker-negative test cells after 24 hours oftreatment in vitro, even though the 24 hour treatment was followed by anovernight recovery period prior to testing the metabolic activity.

FIG. 6B depicts experimental data showing nonspecific cytotoxicity ofthe free PE therapeutic agent on marker-positive and -negative testcells after 24 hours of treatment in vitro.

FIG. 7A graphically presents experimental data showing that a short 5minute “pulse” treatment, i.e., instead of 24 hours, followed byexposure to [3H]leucine, with free RA therapeutic agent beingnonspecifically cytotoxic, i.e., for control HT29 marker-negative cells,but, in contrast, the RA-NR-AN-01 therapeutic conjugate is not cytotoxicin this “pulse” treatment.

FIG. 7B presents graphically experimental data showing that free RAtherapeutic agent is nonspecifically cytotoxic for control HT29marker-negative cells, even in a 5′ “pulse” treatment followed by a 24hour recovery period prior to [3H]leucine exposure, but, in contrast,the RA-NR-AN-01 therapeutic conjugate is not cytotoxic to cells.

FIG. 7C presents graphically results of experiments showing that “pulse”treatment of cells in vitro with the RA-NR-AN-01 therapeutic conjugateinhibits cellular activity in marker-positive A375 cells, as measured byprotein synthesis.

FIG. 7D presents graphically experimental data showing that “pulse”treatment of cells in vitro with the RA-NR-AN-01 therapeutic conjugatedid not exert long-lasting inhibitory effects on cellular activity inmarker-positive cells, since protein synthesis in A375 cells was notinhibited when the cells were allowed an overnight recovery period priorto testing in vitro.

FIG. 8A presents graphically experimental data showing that while a“pulse” treatment of cells in vitro with free RA therapeutic agent wasnon-specifically cytotoxic, the RA-NR-AN-01 therapeutic conjugate didnot exert long-lasting inhibitory effects on cellular activity invascular smooth muscle cells, as evidenced by metabolic activity in BO54cells that were allowed a 48 hour recovery period prior to testing.

FIG. 8B graphically depicts experimental data similar to those presentedin FIG. 8A, above, but using a second marker-positive cell type, namelyA375, the data show that “pulse” treatment with the RA-NR-AN-01therapeutic conjugate did not exert long-lasting inhibitory effects oncellular activity, as measured by metabolic activity in A375 cells thatwere allowed a 48 hour recovery period prior to testing.

FIG. 8C graphically depicts results similar to those presented in FIG.8A and FIG. 8B, above, but using a marker-negative control cell type,namely HT29. The results show that the “pulse” treatment with theRA-NR-AN-01 therapeutic conjugate did not exert long-lasting inhibitoryeffects on the cellular activity of marker-negative control cells, asmeasured by metabolic activity in HT29 cells that were allowed a 48 hourrecovery period prior to testing.

FIG. 9A shows stenosis due to intimal smooth muscle cell proliferationin a histological section of an untreated artery 5 weeks afterangioplasty in an animal model.

FIG. 9B shows inhibition of stenosis in a histological section of anartery treated with therapeutic conjugate at 5 weeks after angioplastyin an animal model.

FIG. 10A graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of suramin withrespect to vascular smooth muscle cells.

FIG. 10B graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of staurosporin withrespect to vascular smooth muscle cells.

FIG. 10C graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of nitroglycerin withrespect to vascular smooth muscle cells.

FIG. 10D graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of cytochalasin B withrespect to vascular smooth muscle cells.

FIG. 11 shows a tangential section parallel to the inner surface of asmooth muscle cell which is magnified 62,500 times and is characterizedby numerous endocytic vesicles, several of which contain antibody coatedgold beads in the process of being internalized by the cell in vitro.

FIG. 12 shows a smooth muscle cell which is magnified 62,500 times andis characterized by a marked accumulation of gold beads in lysosomes at24 hours following exposure of the cell to the beads in vitro.

FIG. 13 shows a smooth muscle cell which is magnified 62,500 times andis characterized by accumulation of gold beads in lysosomes in vivo.

FIG. 14 depicts an in vivo dose response study of the effects ofcytochalasin B on the luminal area of pig femoral arteries.

FIG. 15 is a graph depicting the inhibition of smooth muscle cellproliferation in traumatized vessels over time by cytochalasin B (CB) ortaxol (TAX) administered in silicone wraps (SW).

FIG. 16 is a graph depicting the inhibition of smooth muscle cellproliferation in traumatized vessels over time by 10% or 30% wt/wt CB inSW or 5% wt/wt TAX in SW.

FIG. 17 is a graph depicting the inhibition of smooth muscle cellproliferation in traumatized vessels over time by CB or TAX in silicone,CB in a collagen gel supported by a bovine collagen mesh (CG-CM) or CBin a pluronic gel supported by a bovine collagen mesh (PG-CW).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

“Therapeutic conjugate” means a vascular smooth muscle or aninterstitial matrix binding protein coupled (e.g., optionally through alinker moiety) to a therapeutic agent. Therapeutic conjugates of theinvention are obtained by coupling a vascular smooth muscle bindingprotein to a therapeutic agent. In the therapeutic conjugate, thevascular smooth muscle binding protein performs the function oftargeting the therapeutic conjugate to vascular smooth muscle cells orpericytes, and the therapeutic agent performs the function of inhibitingthe cellular activity or proliferation of the smooth muscle cell orpericyte.

“Therapeutic agent” includes any moiety capable of exerting atherapeutic or prophylactic effect in the present method.

“Target” and “marker” are used interchangeably in describing the presentconjugates to mean a molecule recognized in a specific manner by thematrix or vascular smooth muscle binding protein, e.g., an antigen,polypeptide antigen or cell surface carbohydrate (e.g., a glycolipid,glycoprotein, or proteoglycan) that is expressed on the cell surfacemembranes of a vascular smooth muscle cell or a matrix structure.

“Epitope” is used to refer to a specific site within the “target”molecule that is bound by the matrix or smooth muscle binding protein,e.g., a sequence of three or more amino acids or saccharides.

“Coupled” is used to mean covalent or non-covalent chemical association(i.e., hydrophobic association as through van der Waals forces orcharge-charge interactions) of the matrix or vascular smooth musclebinding protein with the therapeutic agent, including by chelation.Preferably, the binding proteins are associated with the therapeuticagents by means of covalent bonding.

“Linker” means a moiety that couples the matrix or smooth muscle bindingprotein to a therapeutic agent, e.g., as derived from an organicchemical coupling agent.

As used herein, “substantially” pure means at least about 90%,preferably at least about 98%, and more preferably at least about 99%,free of contaminants when assayed by methods conventionally employed bythe art.

As used herein, “substantially” solid or crystalline means at leastabout 90%, preferably at least about 98%, and more preferably at leastabout 99%, free of non-solid or non-crystalline forms or phases whenassayed by methods conventionally employed by the art.

“Migration” of smooth muscle cells means movement of these cells in vivofrom the medial layers of a vessel into the intima, which may also bestudied in vitro by following the motion of a cell from one location toanother (e.g., using time-lapse cinematography or a video recorder andmanual counting of smooth muscle cell migration out of a defined area inthe tissue culture over time).

“Proliferation” means an increase in cell number, i.e., by mitosis ofthe cells. As used herein “smooth muscle cells” does not refer toneoplastic vascular smooth muscle cells, i.e., cancer cells.

“Implantable device” means any material that is capable of retaining andreleasing a therapeutic agent so as to deliver it in situ in acontrolled fashion to a mammalian vessel. An implantable device includesdevices which are placed in the lumen of the vessel, e.g., an indwellingcatheter or stent, or on the exterior of a vessel, e.g., an adventitialwrap, mesh or covering, or which become a part of the vessel itself, forexample to replace a portion of a diseased or traumatized vessel, e.g.,a synthetic graft. The implantable device may comprise the therapeuticagent in a form which is releasably embedded in and/or coated on thedevice. The therapeutic agent may also be releasably embedded in and/orcoated on a pharmaceutically acceptable release carrier matrix, whichmay be applied to and/or embedded in the device or administered directlyto a vessel. The matrix is non-liquid, preferably solid. For example, amatrix useful in the practice of the invention includes, but is notlimited to, a gel, a paste, or a permeable membrane. An implantabledevice may be implanted for a limited amount of time, e.g., catheter orinfusion needle delivery of a therapeutic agent, or for a prolongedperiod of time, e.g., a stent or graft. Vessels, into which theimplantable device of the invention may be inserted, include, but arenot limited to, coronary, femoral, carotid and peripheral vessels.

“Abnormal or pathological or inappropriate” with respect to an activityor proliferation means division, growth or migration of cells, but notcancer cells, that occurs more rapidly or to a significantly greaterextent than typically occurs in a normally functioning cell of the sametype or in lesions not found in healthy tissue.

“Expressed” means mRNA transcription and translation with resultantsynthesis, glycosylation, and/or secretion of a polypeptide by a cell,e.g., CSPG synthesized by a vascular smooth muscle cell or pericyte.

“Vascular remodeling” means a diminution in vessel lumen volume,diameter or area that is not the result of neointimal thickening orsmooth muscle cell proliferation, and which generally occurs after aprocedural vascular trauma. Thus, a reduction in the area(“constriction”) circumscribed by the internal elastic lamina ormembrane (IEL) without significant amounts of neointimal formation istermed “vascular remodeling.” See Isner, Circ., 89, 2937 (1994). Theluminal cross-sectional area of a vessel can be measured by directplanimetering, e.g., by intravascular ultrasound (IVUS) or at necropsy.As used herein, “vascular remodeling” does not include compensatoryenlargement of a vessel which accompanies neointimal proliferation so asto accomodate the intimal increase. This compensatory enlargement hasalso been referred to as “positive” vascular remodeling.

“Sustained release” means a dosage form designed to release atherapeutic agent therefrom for a time period from about 0.0005 to about180, preferably from about 1-3 to about 150, and more preferably fromabout 30 to about 120, and even more preferably about 3 to about 21,days. Release over a longer time period is also contemplated as“sustained release” in the context of the present invention. Moreover,it is contemplated that the invention can be practiced with a locally orsystemically administered sustained release dosage form.

“Dosage form” includes a formulation comprising a free (non-targeted ornon-binding partner associated) therapeutic agent, as well as asustained release formulation comprising a therapeutic agent. Forexample, sustained release formulations can comprise microparticles ornanoparticles, biodegradable or non-biodegradable polymeric materials,or any combination thereof, comprising a therapeutic agent dispersedtherein, as well as crystalline forms of the therapeutic agent. Atargeted or binding partner associated dosage form of the inventionincludes a sustained release therapeutic formulation comprisingmicroparticles or nanoparticles, and/or biodegradable ornon-biodegradable polymeric materials. The sustained release dosage formis linked to one or more binding proteins or peptides, so as to delivera therapeutic agent dispersed therein to a target cell population whichbinds to the binding protein or peptide.

“Cytochalasin” includes a fungal metabolite exhibiting an inhibitoryeffect on target cellular metabolism, including prevention ofcontraction or migration of vascular smooth muscle cells. Preferably,cytochalasins inhibit the polymerization of monomeric actin (G-actin) topolymeric form (F-actin), thereby inhibiting cell functions requiringcytoplasmic microfilaments. Cytochalasins typically are derived fromphenylalanine (cytochalasins), tryptophan (chaetoglobosins), or leucine(aspochalasins), resulting in a benzyl, indol-3-yl methyl or isobutylgroup, respectively, at position C-3 of a substitutedperhydroisoindole-1-one moiety (Formula I to II).

The perhydroisoindole moiety in turn contains an 11-, 13- or 14-atomcarbocyclic- or oxygen-containing ring linked to positions C-8 and C-9.All naturally occurring cytochalasins contain a methyl group at C-5; amethyl or methylene group at C-12; and a methyl group at C-14 or C-16.Exemplary cytochalasins include cytochalasin A, cytochalasin B,cytochalasin C, cytochalasin D, cytochalasin E, cytochalasin F,cytochalasin G, cytochalasin H, cytochalasin J, cytochalasin K,cytochalasin L, cytochalasin M, cytochalasin N, cytochalasin 0,cytochalasin P, cytochalasin Q, cytochalasin R, cytochalasin S,chaetoglobosin A, chaetoglobosin B, chaetoglobosin C, chaetoglobosin D,chaetoglobosin E, chaetoglobosin F, chaetoglobosin G, chaetoglobosin J,chaetoglobosin K, deoxaphomin, proxiphomin, protophomin, zygosporin D,zygosporin E, zygosporin F, zygosporin G, aspochalasin B, aspochalasinC, aspochalasin D and the like, as well as functional equivalents andderivatives thereof. Certain cytochalasin derivatives are set forth inJapanese Patent Nos. 72 01,925; 72 14,219; 72 08,533; 72 23,394; 7201924; and 72 04,164. Preferred cytochalasins include cytochalasin A,cytochalasin B and cytochalasin D. Cytochalasin B is used in thisdescription as a typical cytochalasin.

As referred to herein, “taxol” includes taxol as well as functionalanalogs, equivalents or derivatives thereof. For example, derivativesand analogs of taxol include, but are not limited to, taxotere,baccatin, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine,10-deacetyl-7-epitaxol, 7 epitaxol, 10-deacetylbaccatin III,10-deacetylcephaolmannine and analogs or derivatives disclosed inKingston et al. (New Trends in Nat. Prod. Chem., 26, 219 (1986)),Bringli et al. (WO 93/17121), Golik et al. (EPA 639577), Kelly et al.(WO 95/20582), and Cassady and Dourous (eds., In: Anticancer AgentsBased on Natural Product Models, Academic Press, NY (1980)), thedisclosures of which are incorporated by reference herein. Methods forpreparing taxol and numerous analogs and derivatives thereof are wellknown to the art.

“Macrocyclic trichothecene” is intended to mean any one of the group ofstructurally related sesquiterpenoid macrocyclic mycotoxins produced byseveral species of fungi and characterized by the12,13-epoxytrichothec-9-ene basic structure, e.g., verrucarins androridins that are the products of secondary metabolism in the soil fungiMyrothecium verrucaria and Myrothecium roridium.

There are two broad classes of trichothecenes: those that have only acentral sesquiterpenoid structure and those that have an additionalmacrocyclic ring (simple and macrocyclic trichothecenes, respectively).The simple trichothecenes may be subdivided into three groups (i.e.,Group A, B, and C) as described in U.S. Pat. Nos. 4,744,981 and4,906,452 (incorporated herein by reference). Representative examples ofGroup A simple trichothecenes include: scirpene, roridin C,dihydrotrichothecene, scirpen-4, 8-diol, verrucarol, scirpentriol, T-2tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin,deacetylcalonectrin, calonectrin, diacetylverrucarol,4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,7-hydroxydiacetoxyscirpenol, 8-hydroxydiacetoxy-scirpenol (neosolaniol),7,8-dihydroxydiacetoxyscirpenol, 7-hydroxy-8-acetyldiacetoxyscirpenol,8-acetylneosolaniol, NT-1, NT-2, HT-2, T-2, and acetyl T-2 toxin.Representative examples of Group B simple trichothecenes include:trichothecolone, trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, nivalenol,4-acetylnivalenol (fusarenon-X), 4,15-idacetylnivalenol,4,7,15-triacetylnivalenol, and tetra-acetylnivalenol. Representativeexamples of Group C simple trichothecenes include: crotocol andcrotocin. Representative macrocyclic trichothecenes include verrucarinA, verrucarin B, verrucarin J (Satratoxin C), roridin A, roridin D,roridin E (satratoxin D), roridin H, satratoxin F, satratoxin G,satratoxin H, vertisporin, mytoxin A, mytoxin C, mytoxin B, myrotoxin A,myrotoxin B, myrotoxin C, myrotoxin D, roritoxin A, roritoxin B, androritoxin D. In addition, the general “trichothecene” sesquiterpenoidring structure is also present in compounds termed “baccharins” isolatedfrom the higher plant Baccharis megapotamica, and these are described inthe literature, for instance as disclosed by Jarvis et al. (Chemistry ofAlleopathy, ACS Symposium Series No. 268: ed. A. C. Thompson, 1984, pp.149-159) and Jarvis & Mazzola (Acc. Chem. Res. 15:338-395, 1982)).Trichothecenes are also produced by soil fungi of the class Fungiimperfecti (Bamburg, J. R. Proc. Molec. Subcell. Biol. 8:41-110, 1983)).

“Staurosporin” includes staurosporin, a protein kinase C inhibitor ofthe following formula (III),

as well as diindoloalkaloids having one of the following generalstructures:

More specifically, the term “staurosporin” includes K-252 (see, forexample, Japanese Patent Application No. 62,164,626), BMY-41950 (U.S.Pat. No. 5,015,578), UCN-01 (U.S. Pat. No. 4,935,415), TAN-999 (JapanesePatent Application No. 01,149,791), TAN-1030A (Japanese PatentApplication No. 01,246,288), RK-286C (Japanese Patent Application No.02,258,724) and functional equivalents and derivatives thereof.Derivatives of staurosporin include those discussed in Japanese PatentApplication Nos. 03,72,485; 01,143,877; 02,09,819 and 03,220,194, aswell as in PCT International Application Nos. WO 89 07,105 and WO 9109,034 and European Patent Application Nos. EP 410,389 and EP 296,110.Derivatives of K-252, a natural product, are known. See, for example,Japanese Patent Application Nos. 63,295,988; 62,240,689; 61,268,687;62,155,284; 62,155,285; 62,120,388 and 63,295,589, as well as PCTInternational Application No. WO 88 07,045 and European PatentApplication No. EP 323,171.

As referred to herein, smooth muscle cells and pericytes include thosecells derived from the medial layers of vessels and adventitial vesselswhich proliferate in intimal hyperplastic vascular sites followinginjury, such as that caused during PTCA. Characteristics of smoothmuscle cells include a histological morphology (under light microscopicexamination) of a spindle shape with an oblong nucleus located centrallyin the cell with nucleoli present and myofibrils in the sarcoplasm.Under electron microscopic examination, smooth muscle cells have longslender mitochondria in the juxtanuclear sarcoplasm, a few tubularelements of granular endoplasmic reticulum, and numerous clusters offree ribosomes. A small Golgi complex may also be located near one poleof the nucleus. The majority of the sarcoplasm is occupied by thin,parallel myofilaments that may be, for the most part, oriented to thelong axis of the muscle cell. These actin containing myofibrils may bearranged in bundles with mitochondria interspersed among them. Scatteredthrough the contractile substance of the cell may also be oval denseareas, with similar dense areas distributed at intervals along the inneraspects of the plasmalemma.

Characteristics of pericytes include a histological morphology (underlight microscopic examination) characterized by an irregular cell shape.Pericytes are found within the basement membrane that surrounds vascularendothelial cells and their identity may be confirmed by positiveimmuno-staining with antibodies specific for alpha smooth muscle actin(e.g., anti-alpha-sm1, Biomakor, Rehovot, Israel), HMW-MAA, and pericyteganglioside antigens e.g., MAb 3G5 (Schlingemann et al., Am. J. Pathol.,136: 1393-1405 (1990)); and, negative immuno-staining with antibodies tocytokeratins (i.e., epithelial and fibroblast markers) and vonWilldebrand factor (i.e., an endothelial marker). Both vascular smoothmuscle cells and pericytes are positive by immunostaining with theNR-AN-01 monoclonal antibody.

As used herein, the term “procedural vascular trauma” includes theeffects of surgical/mechanical interventions into mammalian vasculature,but does not include vascular trauma due to the organic vascularpathologies, i.e., diseases and infections. Thus, procedural vasculartraumas within the scope of the present treatment method include (1)organ transplantation, such as heart, kidney, liver and the like, e.g.,involving vessel anastomosis; (2) vascular surgery, e.g., coronarybypass surgery, biopsy, heart valve replacement, atheroectomy,thrombectomy, and the like; (3) transcatheter vascular therapies (TVT)including angioplasty, e.g., laser angioplasty and PTCA procedures,employing balloon catheters, and indwelling catheters; (4) vasculargrafting using natural or synthetic materials, such as in saphenous veincoronary bypass grafts, dacron and venous grafts used for peripheralarterial reconstruction, etc.; (5) placement of a mechanical shunt,e.g., a PTFE hemodialysis shunt used for arteriovenous communications;and (6) placement of an intravascular stent, which may be metallic,plastic or a biodegradable polymer. See U.S. patent application Ser. No.08/389,712, filed Feb. 15, 1995, which is incorporated by referenceherein. For a general discussion of implantable devices and biomaterialsfrom which they can be formed, see H. Kambic et al., “Biomaterials inArtificial Organs”, Chem. Eng. News, 30 (Apr. 14, 1986), the disclosureof which is incorporated by reference herein.

Therapeutic Agents Falling Within the Scope of the Invention

Therapeutic agents useful in the practice of the invention includeagents which biologically stent a vessel and/or reduce or inhibitvascular remodeling and/or inhibit or reduce vascular smooth muscle cellproliferation following a procedural vascular trauma. The therapeuticagents of the invention are selected to inhibit a cellular activity of avascular smooth muscle cell, e.g., proliferation, migration, increase incell volume, increase in extracellular matrix synthesis (e.g.,collagens, proteoglycans, and the like), or secretion of extracellularmatrix materials by the cell.

Preferably, the therapeutic agent is: a) a “cytostatic agent” which actsto prevent or delay cell division in proliferating cells by inhibitingreplication of DNA or by inhibiting spindle fiber formation and thelike; b) an inhibitor of migration of vascular smooth muscle cells fromthe medial wall into the intima, e.g., an “anti-migratory agent” e.g., acytochalasin; c) as an inhibitor of the intracellular increase in cellvolume (i.e., the tissue volume occupied by a cell; a “cytoskeletalinhibitor” or a “metabolic inhibitor”); d) an inhibitor that blockscellular protein synthesis and/or secretion or organization ofextracellular matrix (i.e., an “anti-matrix agent”); or any combinationthereof.

Representative examples of “cytostatic agents” include, e.g., modifiedtoxins, methotrexate, adriamycin, radionuclides (e.g., see Fritzberg etal., U.S. Pat. No. 4,897,255), protein kinase inhibitors (e.g.,staurosporin), taxol or analogs thereof (e.g., taxotere), inhibitors ofspecific enzymes (such as the nuclear enzyme DNA topoisomerase II andDNA polymerase, RNA polymerase, adenyl guanyl cyclase), superoxidedismutase inhibitors, terminal deoxynucleotidyl-transferase, reversetranscriptase, antisense oligonucleotides that suppress smooth musclecell proliferation and the like, which when delivered into a cellularcompartment at an appropriate dosage will act to impair proliferation ofa smooth muscle cell or pericyte without killing the cell.

Other examples of “cytostatic agents” include peptidic or mimeticinhibitors (i.e., antagonists, agonists, or competitive ornon-competitive inhibitors) of cellular factors that may (e.g., in thepresence of extracellular matrix) trigger proliferation of smooth musclecells or pericytes: e.g., cytokines (e.g., interleukins such as IL-1),growth factors, (e.g., PDGF, TGF-alpha or -beta, tumor necrosis factor,smooth muscle- and endothelial-derived growth factors, i.e., endothelin,FGF), homing receptors (e.g., for platelets or leukocytes), andextracellular matrix receptors (e.g., integrins). Representativeexamples of useful therapeutic agents in this category of cytostaticagents for smooth muscle proliferation include: subfragments of heparin,triazolopyrimidine (Trapidil; a PDGF antagonist), lovastatin, andprostaglandins E1 or I2.

Representative examples of “anti-migratory agents” include inhibitors(i.e., agonists and antagonists, and competitive or non-competitiveinhibitors) of chemotactic factors and their receptors (e.g., complementchemotaxins such as C5a, C5a desarg or C4a; extracellular matrixfactors, e.g., collagen degradation fragments), or of intracellularcytoskeletal proteins involved in locomotion (e.g., actin, cytoskeletalelements, and phosphatases and kinases involved in locomotion).Representative examples of useful therapeutic agents in this category ofanti-migratory agents include caffeic acid derivatives and nilvadipine(a calcium antagonist), and steroid hormones. Preferred anti-migratorytherapeutic agents are the cytochalasins.

Representative examples of “cytoskeletal inhibitors”, a subset ofcytostatic agents, include colchicine, vinblastin, cytochalasins, taxol,or analogs or derivatives thereof that act on microtubule andmicrofilament networks within a cell. Preferred cytoskeletal inhibitorsinclude cytochalasin B, cytochalasin A, cytochalasin D and taxol.

Representative examples of “anti-matrix agents” include inhibitors(i.e., agonists and antagonists and competitive and non-competitiveinhibitors) of matrix synthesis, secretion and assembly, organizationalcross-linking (e.g., transglutaminases cross-linking collagen), andmatrix remodeling (e.g., following wound healing). A representativeexample of a useful therapeutic agent in this category of anti-matrixagents is colchicine, an inhibitor of secretion of extracellular matrix.Another example is tamoxifen for which evidence exists regarding itscapability to organize and/or stabilize as well as diminish smoothmuscle cell proliferation following angioplasty. The organization orstabilization may stem from the blockage of vascular smooth muscle cellmaturation into a pathologically proliferating form.

Representative examples of “metabolic inhibitors” include staurosporin,trichothecenes, and modified diphtheria and ricin toxins, Pseudomonasexotoxin and the like. In a preferred embodiment, the therapeuticconjugate is constructed with a therapeutic agent that is a simpletrichothecene or a macrocyclic trichothecene, e.g., a verrucarin orroridin. Trichothecenes are drugs produced by soil fungi of the classFungi imperfecti or isolated from Baccharus megapotamica (Bamburg, J. R.Proc. Molec. Subcell. Biol. 8:41-110, 1983; Jarvis & Mazzola, Acc. Chem.Res. 15:338-395, 1982). They appear to be the most toxic molecules thatcontain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org.Naturst. 31:61-117, 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation,elongation, or termination phases.

Identification of Therapeutic Agents Useful in the Practice of theInvention

The identification of therapeutic agents useful in the practice of theinvention may be determined by methods well known to the art. Forexample, a therapeutic agent falling within the scope of the inventionexhibits one or more of the following characteristics. The agent:

(i) results in retention of an expanded luminal cross-sectional area,diameter or volume of a vessel following angioplasty (e.g., PTCA,percutaneous transluminal angioplasty (PTA) or the like) or othertrauma, including atheroectomy (e.g., rotoblater, laser and the like),coronary artery bypass procedures and the like; or resulting fromvascular disease, e.g, atherosclerosis, eye diseases secondary tovascular stenosis or atrphy, cerebral vascular stenotic diseases or thelike;

(ii) facilitates an initial increase in luminal cross-sectional area,diameter or volume that does not result in or accentuate chronicstenosis of the lumen;

(iii) inhibits target cell contraction or migration; and

(iv) is cytostatic.

Methods to measure luminal cross-sectional area, volume or diameterinclude, but are not limited to, angiography, ultrasonic evaluation,fluoroscopic imaging, fiber optic endoscopic examination or biopsy andhistology.

Preferably, a therapeutic agent employed herein will have all fourproperties; however, the first and third are generally more importantthan the second and fourth for practice of the present invention. It wasfound that cytochalasin B administration can result in a biologicalstenting effect. The biological stenting effect can be achieved using asingle infusion of the therapeutic agent into the traumatized region ofthe vessel wall at a dose concentration ranging from about 0.1micrograms/ml to about 1.0 micrograms/ml, and preferably about 0.01micrograms/ml to about 10.0 micrograms/ml (Example 16).

In the case of therapeutic agents or dosage forms containinganti-migratory or anti-matrix therapeutic agents, cell migration andcell adherence in in vitro assays, respectively, may be used fordetermining the concentration at which a therapeutically effectivedosage will be reached in the fluid space in the vessel wall created byan infusion catheter.

An agent useful in the sustained release embodiments of the presentinvention exhibits one or more of the following characteristics. Theagent

(i) causes the retention of an expanded luminal diameter orcross-sectional area following angioplasty (e.g., PTCA, percutaneoustransluminal angioplasty (PTA) or the like) or other trauma, includingatheroectomy (e.g., rotoblater, laser and the like), coronary arterybypass procedures or the like; or resulting from vascular disease, e.g,atherosclerosis, eye diseases secondary to vascular stenosis or atrphy,cerebral vascular stenotic diseases or the like;

(ii) inhibits target cell proliferation (e.g., following 5 minute and 24hour exposure to the agent, in vitro vascular smooth muscle tissuecultures demonstrate a level of inhibition of ³H-thymidine uptake and,preferably, display relatively less inhibition of ³H-leucine uptake);

(iii) at a dose sufficient to inhibit DNA synthesis, produces only mildto moderate (e.g., grade 2 or 3 in the assays described below)morphological cytotoxic effects;

(iv) inhibits target cell contraction; and

(v) is cytostatic.

Upon identification of a therapeutic agent exhibiting one or more of thepreceding properties, the agent is subjected to a second testingprotocol that involves longer exposure of vascular smooth muscle cells(VSMC) to the therapeutic agent. For example, an agent useful in thesustained release embodiments of the present invention also exhibits thefollowing characteristics:

(i) upon long term (e.g., 5 days) exposure, the agent produces the sameor similar in vitro effect on vascular smooth muscle tissue culture DNAsynthesis and protein synthesis, as described above for the 5 minute and24 hour exposures; and

(ii) at an effective dose in the long term in vitro assay for DNAsynthesis inhibition, the agent exhibits mild to moderate morphologicalcytotoxic effects over a longer term (e.g., 10 days).

Further evaluation of potentially useful anti-proliferative agents isconducted in an in vivo balloon traumatized pig femoral artery model.Preferably, these agents demonstrate a 50% or greater inhibition of cellproliferation in the tunica media vascular smooth muscle cells, asindicated by a 1 hour “BRDU flash labeling” prior to tissue collectionand histological evaluation (Example 13). If an agent is effective inthis assay to inhibit intimal smooth muscle proliferation by 50% or morewith a single exposure, it does not require administration in asustained release dosage form.

Agents are evaluated for sustained release if the systemic toxicity andpotential therapeutic index appear to permit intravenous administrationto achieve the 50% inhibition threshold, or if the agent is amenable tolocal delivery to the vascular smooth muscle cells via sustained releaseat an effective anti-proliferative dose. Agents are evaluated in asustained release dosage form for dose optimization and efficacystudies. Preferably, anti-proliferative agents useful in the practice ofthe present invention decrease vascular stenosis by 50% in balloontraumatized pig femoral arteries and, more preferably, decrease vascularstenosis to a similar extent in pig coronary arteries.

Inhibition of cellular proliferation (i.e., DNA synthesis) is theprimary characteristic of agents useful in sustained release dosageforms. For example, a preferred therapeutic agent exhibits adifferential between ³H-leucine and ³H-thymidine uptake so that it canbe administered at cytostatic doses. Moreover, cytotoxicity studiesshould indicate that prolonged exposure to the therapeutic agent wouldnot adversely impact the target cells. In addition, BRDU pulsing shouldindicate that the therapeutic agent is effective to inhibit target cellproliferation. Any convenient method for evaluating the capability of anagent to inhibit cell proliferation may alternatively be employed,however.

Sustained Released Dosage Forms

Sustained release dosage forms of the invention may comprisemicroparticles and/or nanoparticles having a therapeutic agent dispersedtherein or may comprise the therapeutic agent in pure, preferablycrystalline, solid form. For sustained release administration,microparticle dosage forms comprising pure, preferably crystalline,therapeutic agents are preferred. The therapeutic dosage forms of thisaspect of the present invention may be of any configuration suitable forsustained release. Preferred sustained release therapeutic dosage formsexhibit one or more of the following characteristics:

microparticles (e.g., from about 0.5 micrometers to about 100micrometers in diameter, with about 0.5 to about 2 micrometers morepreferred; or from about 0.01 micrometers to about 200 micrometers indiameter, preferably from about 0.5 to about 50 micrometers, and morepreferably from about 2 to about 15 micrometers) or nanoparticles (e.g.,from about 1.0 nanometer to about 1000 nanometers in diameter, withabout 50 to about 250 nanometers being more preferred; or from about0.01 nanometer to about 1000 nanometers in diameter, preferably fromabout 50 to about 200 nanometers), free flowing powder structure;

biodegradable structure designed to biodegrade over a period of timepreferably between from about 0.5 to about 180 days, preferably fromabout 1-3 to about 150 days, or from about 3 to about 180 days, withfrom about 10 to about 21 days more preferred; or non-biodegradablestructure to allow therapeutic agent diffusion to occur over a timeperiod of between from about 0.5 to about 180 days, more preferably fromabout 30 to about 120 days; or from about 3 to about 180 days, with fromabout 10 to about 21 days preferred;

biocompatible with target tissue and the local physiological environmentinto which the dosage form to be administered, including yieldingbiocompatible biodegradation products;

facilitate a stable and reproducible dispersion of therapeutic agenttherein, preferably to form a therapeutic agent-polymer matrix, withactive therapeutic agent release occurring by one or both of thefollowing routes: (1) diffusion of the therapeutic agent through thedosage form (when the therapeutic agent is soluble in the shaped polymeror polymer mixture defining the dimensions of the dosage form); or (2)release of the therapeutic agent as the dosage form biodegrades; and/or

for targeted dosage forms, capability to have, preferably, from about 1to about 10,000 binding protein/peptide to dosage form bonds and morepreferably, a maximum of about 1 binding peptide to dosage form bond per150 square angstroms of particle surface area. The total number ofbinding protein/peptide to dosage form bonds depends upon the particlesize used. The binding proteins or peptides are capable of coupling tothe particles of the therapeutic dosage form through covalent ligandsandwich or non-covalent modalities as set forth herein.

Nanoparticle sustained release therapeutic dosage forms are preferablybiodegradable and, optionally, bind to the vascular smooth muscle cellsand enter those cells, primarily by endocytosis. The biodegradation ofthe nanoparticles occurs over time (e.g., 30 to 120 days; or 10 to 21days) in prelysosomic vesicles and lysosomes. Preferred largermicroparticle therapeutic dosage forms of the present invention releasethe therapeutic agents for subsequent target cell uptake with only a fewof the smaller microparticles entering the cell by phagocytosis. Apractitioner in the art will appreciate that the precise mechanism bywhich a target cell assimilates and metabolizes a dosage form of thepresent invention depends on the morphology, physiology and metabolicprocesses of those cells. The size of the article sustained releasetherapeutic dosage forms is also important with respect to the mode ofcellular assimilation. For example, the smaller nanoparticles can flowwith the interstitial fluid between cells and penetrate the infusedtissue. The larger microparticles tend to be more easily trappedinterstitially in the infused primary tissue, and thus are useful todeliver anti-proliferative therapeutic agents.

Preferred sustained release dosage forms of the present inventioncomprise biodegradable microparticles or nanoparticles. More preferably,biodegradable microparticles or nanoparticles are formed of a polymercontaining matrix that biodegrades by random, nonenzymatic, hydrolyticscissioning to release therapeutic agent, thereby forming pores withinthe particulate structure.

Polymers derived from the condensation of alpha hydroxycarboxylic acidsand related lactones are preferred for use in the present invention. Aparticularly preferred moiety is formed of a mixture of thermoplasticpolyesters (e.g., polylactide or polyglycolide) or a copolymer oflactide and glycolide components, such as poly(lactide-co-glycolide). Anexemplary structure, a random poly(DL-lactide-co-glycolide), is shownbelow, with the values of x and y being manipulable by a practitioner inthe art to achieve desirable microparticle or nanoparticle properties.

Other agents suitable for forming particulate dosage forms of thepresent invention include polyorthoesters and polyacetals (PolymerLetters, 18:293 (1980) and polyorthocarbonates (U.S. Pat. No. 4,093,709)and the like.

Preferred lactic acid/glycolic acid polymer containing matrix particlesof the present invention are prepared by emulsion-based processes, thatconstitute modified solvent extraction processes, see, for example,processes described by Cowsar et al., “Poly(Lactide-Co-Glycolide)Microcapsules for Controlled Release of Steroids,” Methods Enzymology,112:101-116, 1985 (steroid entrapment in microparticles); Eldridge etal., “Biodegradable and Biocompatible Poly(DL-Lactide-Co-Glycolide)Microspheres as an Adjuvant for Staphylococcal Enterotoxin B ToxoidWhich Enhances the Level of Toxin-Neutralizing Antibodies,” Infectionand Immunity, 59:2978-2986, 1991 (toxoid entrapment); Cohen et al.,“Controlled Delivery Systems for Proteins Based on Poly(Lactic/GlycolicAcid) Microspheres,” Pharmaceutical Research, 8(6):713-720, 1991 (enzymeentrapment); and Sanders et al., “Controlled Release of a LuteinizingHormone-Releasing Hormone Analogue from Poly(D,L-Lactide-Co-Glycolide)Microspheres,” J. Pharmaceutical Science, 73(9):1294-1297, 1984 (peptideentrapment).

In general, the procedure for forming particle dosage forms of thepresent invention involves dissolving the polymer in a halogenatedhydrocarbon solvent, dispersing a therapeutic agent solution (preferablyaqueous) therein, and adding an additional agent that acts as a solventfor the halogenated hydrocarbon solvent but not for the polymer. Thepolymer precipitates out from the polymer-halogenated hydrocarbonsolution onto droplets of the therapeutic agent containing solution andentraps the therapeutic agent. Preferably the therapeutic agent issubstantially uniformly dispersed within the sustained release dosageform of the present invention. Following particle formation, they arewashed and hardened with an organic solvent. Water washing and aqueousnonionic surfactant washing steps follow, prior to drying at roomtemperature under vacuum.

For biocompatibility purposes, particulate dosage forms, characterizedby a therapeutic agent dispersed in the matrix of the particles, aresterilized prior to packaging, storage or administration. Sterilizationmay be conducted in any convenient manner therefor. For example, theparticles can be irradiated with gamma radiation, provided that exposureto such radiation does not adversely impact the structure or function ofthe therapeutic agent dispersed in the therapeutic agent-polymer matrixor the binding protein/peptide attached thereto. If the therapeuticagent or binding protein/peptide is so adversely impacted, the particledosage forms can be produced under sterile conditions.

Release of the therapeutic agent from the particle dosage forms of thepresent invention can occur as a result of both diffusion and particlematrix erosion. The biodegradation rate directly effects the kinetics oftherapeutic agent release. The biodegradation rate is regulable byalteration of the composition or structure of the sustained releasedosage form. For example, alteration of the lactide/glycolide ratio inpreferred dosage forms of the present invention can be conducted, asdescribed by Tice et al., “Biodegradable Controlled-Release ParenteralSystems,” Pharmaceutical Technology, pp. 26-35, 1984; by inclusion ofagents that alter the rate of polymer hydrolysis, such as citric acidand sodium carbonate, as described by Kent et al., “Microencapsulationof Water Soluble Active Polypeptides,” U.S. Pat. No. 4,675,189; byaltering the loading of therapeutic agent in the lactide/glycolidepolymer, the degradation rate being inversely proportional to the amountof therapeutic agent contained therein, by judicious selection of anappropriate analog of a common family of therapeutic agents that exhibitdifferent potencies so as to alter said core loadings; and by variationof particle size, as described by Beck et al.,“Poly(DL-Lactide-Co-Glycolide)/Norethisterone Microcapsules: AnInjectable Biodegradable Contraceptive,” Biol. Reprod., 28:186-195,1983, or the like. All of the aforementioned methods of regulatingbiodegradation rate influence the intrinsic viscosity of the polymercontaining matrix, thereby altering the hydration rate thereof.

The preferred lactide/glycolide structure is biocompatible with themammalian physiological environment. Also, these preferred sustainedrelease dosage forms have the advantage that biodegradation thereofforms lactic acid and glycolic acid, both normal metabolic products ofmammals.

Functional groups required for binding of the protein/peptide to theparticle dosage form are optionally included in or on the particlematrix and are attached to the non-degradable or biodegradable polymericunits. Functional groups that are useful for this purpose include thosethat are reactive with peptides, e.g., carboxyl groups, amine groups,sulfhydryl groups and the like. Preferred binding enhancement moietiesinclude the terminal carboxyl groups of the preferred(lactide-glycolide) polymer containing matrix or the like.

Therapeutic agents useful in the sustained release dosage forms of thepresent invention preferably are those that inhibit vascular smoothmuscle cell activity without killing the cells (i.e., cytostatictherapeutic agents). A cytostatic agent can also be defined as a moietycapable of inhibiting one or more pathological activities of the targetcells for a time sufficient to achieve a therapeutic benefit. Preferredtherapeutic agents thus exhibit one or more of the followingcapabilities: inhibition of DNA synthesis prior to protein synthesisinhibition, or inhibition of migration of vascular smooth muscle cellsinto the intima. These therapeutic agents do not significantly inhibitprotein synthesis (i.e., do not kill the target cells) and, therefore,facilitate cellular repair and matrix production, which in turn acts tostabilize the vascular wall lesion caused by angioplasty, by reducingsmooth muscle cell proliferation.

Preferred therapeutic agents are protein kinase inhibitors, such asstaurosporin (staurosporine is available from Sigma Chemical Co., St.Louis, Mo.), and cytoskeletal inhibitors such as the cytochalasins,e.g., cytochalasin B (Sigma Chemical Co.), nitroglycerin (DuPontPharmaceuticals, Inc., Mauti, Puerto Rico) and taxol, or analogs orfunctional equivalents thereof. These compounds are cytostatic and havebeen shown to exert minimal protein synthesis inhibition andcytotoxicity at concentrations at which significant DNA synthesisinhibition occurs (see Example 8 and FIGS. 10A-10D). A useful protocolfor identifying therapeutic agents useful in sustained release dosageform embodiments of the present invention is set forth in Example 8, forexample.

To prepare one embodiment of the invention, a cytoskeletal inhibitor,e.g., cytochalasin B, is incorporated into biodegradable poly(DL-lactide-co-glycolide) microparticles or into nanoparticles. Themicroparticles are about 1 to about 50μ, preferably 4μ to about 15μ, andmore preferably about 2 to about 15μ, in diameter. The nanoparticles areabout 5 to about 500 nanometers, preferably about 10 to about 250nanometers, and more preferably about 50 to about 200 nanometers, indiameter. The microparticles or nanoparticles comprising the therapeuticagent can be further embedded in or on an implantable device, e.g., in astent coating, or delivered in a suitable liquid vehicle by animplantable device, e.g., via an infusion catheter. Preferably, thesustained release dosage form is biodegradable and, preferably,biodegrades over about 30-120 days. The sustained release dosage form ispreferably administered during the procedural vascular trauma.

A preferred sustained release dosage form of the invention comprisesbiodegradable microparticles, preferably about 2 to about 15μ indiameter, which are tissue compatible and physically compatible with animplantable device, e.g., a needle infusion catheter or a microinfusioncatheter. Another preferred sustained release dosage form of theinvention comprises biodegradable nanoparticles, preferably about 50 toabout 200 nanometers in diameter, which are tissue compatible andphysically compatible with an implantable device, e.g., a needleinfusion catheter or a microinfusion catheter. To deliver the sustainedrelease dosage forms by balloon catheter, the balloon pore or hole sizesare preferably about 0.1 to about 8 micron, more preferably about 0.2 toabout 0.8 micron, in diameter.

The cellular concentration of the cytoskeletal inhibitor that isattained in the tunica media and/or intima of the treated vessel iseffective to inhibit vascular smooth muscle cell proliferation andmigration, e.g., a cellular concentration at least about 0.1 μg/mlcytochalasin B is attained. The inhibition of the smooth muscle cellsresults in a more rapid and complete re-endothelialization after aprocedural vascular trauma, e.g., intraventional placement of the stent.The increased rate of re-endothelialization reduces loss in luminalcross-sectional area or diameter and reduces decreases in blood flow.

Another preferred sustained release dosage form of the inventioncomprises a pure, solid crystalline form of a therapeutic agent,preferably, of a cytoskeletal inhibitor. This embodiment of thesustained release dosage form of the present invention preferablyfurther comprises a tissue-compatible pharmaceutically acceptable matrixcarrier that provides a supporting structure for the crystals, e.g., ashaped body of silicone, collagen gel retained in a collagen mesh,pluronic gel retained in a collagen mesh, or mannitol retained in ashaped body of silicone. Thus, for example, sustained release dosageforms comprising cytochalasin B and a pharmaceutical matrix carrierpreferably comprise about 5 to about 70%, more preferably about 7 toabout 40%, and even more preferably about 5 to about 30%, weight percentof cytochalasin B/weight percent of the total matrix carrier-therapeuticagent sustained release dosage form. Sustained release dosage formscomprising taxol and a pharmaceutical matrix carrier preferably compriseabout 1 to about 70%, more preferably about 2 to about 50%, and evenmore preferably about 3 to about 8%, weight percent of taxol/weightpercent of the total matrix carrier-therapeutic agent sustained releasedosage form.

Identification and Preparation of Targeted Dosage Forms Useful in thePractice of the Invention

Vascular smooth muscle cell binding proteins useful in the inventionbind to targets on the surface of vascular smooth muscle cells. A usefulvascular smooth muscle binding protein is a polypeptide, peptidic, ormimetic compound (as described below) that is capable of binding to atarget or marker on a surface component of an intact or disruptedvascular smooth muscle cell. Such binding allows for either release oftherapeutic agent extracellularly in the immediate interstitial matrixwith subsequent diffusion of therapeutic agent into the remaining intactsmooth muscle cells and/or internalization by the cell into anintracellular compartment of the entire targeted biodegradable moiety,thus permitting delivery of the therapeutic agent thereto. It will berecognized that specific targets, e.g., polypeptides or carbohydrates,proteoglycans and the like, that are associated with the cell membranesof vascular smooth muscle cells are useful for selecting (e.g., bycloning) or constructing (e.g., by genetic engineering or chemicalsynthesis) appropriately specific vascular smooth muscle bindingproteins. Particularly useful “targets” are internalized by smoothmuscle cells, e.g., as membrane constituent antigen turnover occurs inrenewal. Internalization by cells may also occur by mechanisms involvingphagolysosomes, clathrin-coated pits, receptor-mediated redistributionor endocytosis and the like.

Representative examples of useful vascular smooth muscle bindingproteins include antibodies (e.g., monoclonal and polyclonalantibodies), F(ab′)₂, Fab′, Fab, and Fv fragments and/or complementaritydetermining regions (CDR) of those antibodies or functional equivalentsthereof, (e.g., binding peptides and the like)); growth factors,cytokines, and polypeptide hormones and the like; and macromoleculesrecognizing extracellular matrix receptors (e.g., integrin andfibronectin receptors and the like).

In a preferred embodiment, e.g., a “target” is exemplified bychondroitin sulfate proteoglycans (CSPGs) synthesized by vascular smoothmuscle cells and pericytes, and a discrete portion (termed an epitopeherein) of the CSPG molecule having an apparent molecular weight ofabout 250 kD is especially preferred as a target. The 250 kD target isan N-linked glycoprotein that is a component of a larger 400 kDproteoglycan complex (Bumol et al., PNAS USA, 79: 1245-1249 (1982)). Inone presently preferred embodiment of the invention, a vascular smoothmuscle binding protein is provided by the NR-AN-01 monoclonal antibody(a subculture of NR-ML-05) that binds to an epitope in a vascular smoothmuscle CSPG target molecule. The monoclonal antibody designated NR-ML-05reportedly binds a 250 kD CSPG synthesized by melanoma cells (Morgan etal., U.S. Pat. No. 4,897,255).

Smooth muscle cells and pericytes also reportedly synthesize a 250 kDCSPG as well as other CSPGs (Schlingeman et al., supra). NR-ML-05binding to smooth muscle cells has been disclosed (Fritzberg et al.,U.S. Pat. No. 4,879,225). The hybridoma, NR-ML-05, which secretes amonoclonal antibody which binds to the 400 kD CSPG, has been depositedwith the American Type Culture Collection, Rockville, Md. and grantedAccession No. 9350. NR-ML-05 is the parent of, and structurally andfunctionally equivalent to, subclone NR-AN-01, disclosed herein.

It will be recognized that NR-AN-01 is just one example of a vascularsmooth muscle binding protein that specifically associates with the 400kD CSPG target, and that other binding proteins associating with thistarget and other epitopes on this target (Bumol et al., PNAS USA, 79:1245-1249 (1982)) are also useful in the therapeutic conjugates andmethods of the invention.

Six other murine monoclonal antibodies and two human chimeric monoclonalantibodies have also been selected, as described herein, thatspecifically target to the 250 kD CSPG of vascular smooth muscle cells.The antibodies also appear to be internalized by the smooth muscle cellsfollowing binding to the cell membrane. Immunoreactivity studies havealso shown the binding of the murine MAbs to the 250 kD antigen in 45human normal tissues and 30 different neoplasms and some of theseresults have been disclosed previously (U.S. Pat. No. 4,879,225). Inthis disclosure and other human clinical studies, MAbs directed to theCSPG 250 kD antigen localized to vascular smooth muscle cells in vivo.Further, it will be recognized that the amino acid residues involved inthe multi-point kinetic association of the NR-AN-01 monoclonal antibodywith a CSPG marker antigenic epitope (i.e., the amino acids constitutingthe complementarity determining regions) are determined bycomputer-assisted molecular modeling and by the use of mutants havingaltered antibody binding affinity. The binding-site amino acids andthree dimensional model of the NR-AN-01 antigen binding site serve as amolecular model for constructing functional equivalents, e.g., shortpolypeptides (“minimal polypeptides”), that have binding affinity for aCSPG synthesized by vascular smooth muscle cells and pericytes.

For treating stenosis following vascular surgical procedures, e.g.,PTCA, preferred binding proteins, e.g., antibodies or fragments, for usein the practice of the invention have a binding affinity of >10⁴liter/mole for the vascular smooth muscle 250 kD CSPG, and also theability to be bound to and internalized by smooth muscle cells orpericytes.

Further, it will be recognized that the amino acid residues involved inthe multi-point kinetic association of the NR-AN-01 monoclonal antibodywith a CSPG marker antigenic epitope (i.e., the amino acids constitutingthe complementarity determining regions) can be determined bycomputer-assisted molecular modeling and by the use of mutants havingaltered antibody binding affinity. The binding-site amino acids andthree dimensional model of the NR-AN-01 antigen binding site can serveas a molecular model for constructing functional equivalents, e.g.,short polypeptides (“minimal polypeptides”), that have binding affinityfor a CSPG synthesized by vascular smooth muscle cells and pericytes.

Three-dimensional modeling is also useful to construct other functionalequivalents that mimic the binding of NR-AN-01 to its antigenic epitope,e.g., “mimetic” chemical compounds that mimic the three-dimensionalaspects of NR-AN-01 binding to its epitope in a CSPG target antigen. Asused herein, “minimal polypeptide” refers to an amino acid sequence ofat least six amino acids in length. As used herein, the term “mimetic”refers to an organic chemical oligomer or polymer constructed to achievethe proper spacing for binding to the amino acids of, for example, anNR-AN-01 CSPG target synthesized by vascular smooth muscle cells orpericytes.

It is also envisioned that human monoclonal antibodies or “humanized”murine antibodies which bind to a vascular smooth muscle binding proteinare useful in the therapeutic conjugates of their invention. Forexample, murine monoclonal antibody may be “chimerized” by geneticallyrecombining the nucleotide sequence encoding the murine Fv region (i.e.,containing the antigen binding sites) with the nucleotide sequenceencoding a human constant domain region and an Fc region, e.g., in amanner similar to that disclosed in European Patent Application No.411,893. Some murine residues may also be retained within the humanvariable region framework domains to ensure proper target site bindingcharacteristics. Humanized vascular smooth muscle binding partners willbe recognized to have the advantage of decreasing the immunoreactivityof the antibody or polypeptide in the host recipient, and may be usefulfor increasing the in vivo half-life and reducing the possibility ofadverse immune reactions tot he conjugate.

Also contemplated as useful binding peptides for sustained releasedosage forms adapted for restenosis treatment are those that localize tointercellular stroma and matrix located between and among vascularsmooth muscle cells. Such binding peptides can deliver the therapeuticagent to the interstitial space between the target cells. Thetherapeutic agent is released into the interstitial spaces forsubsequent uptake by the vascular smooth muscle cells. Preferred bindingpeptides of this type are associated with epitopes on collagen,extracellular glycoproteins, e.g., tenascin, reticulum and elasticfibers, cytokeratin and other intercellular matrix components. Minimalpeptides, mimetic organic chemical compounds, human or humanizedmonoclonal antibodies and the like that localize to intracellular stromaand matrix are also useful as binding peptides in this embodiment of thepresent invention. These binding peptides may be identified andconstructed or isolated in accordance with known techniques. Inpreferred embodiments of the present invention, the interstitial matrixbinding protein binds to a target epitope with an association constantof at least about 10⁻⁴ M.

Representative “coupling” methods for linking the therapeutic agentthrough covalent or non-covalent bonds to the vascular smooth musclebinding protein include chemical cross-linkers and heterobifunctionalcross-linking compounds (i.e., “linkers”) that react to form a bondbetween reactive groups (such as hydroxyl, amino, amido, or sulfhydrylgroups) in a therapeutic agent and other reactive groups (of a similarnature) in the vascular smooth muscle binding protein. This bond may be,for example, a peptide bond, disulfide bond, thioester bond, amide bond,thioether bond, and the like.

In one illustrative example, conjugates of monoclonal antibodies withdrugs have been summarized by Morgan and Foon (Monoclonal AntibodyTherapy to Cancer: Preclinical Models and Investigations, Basic andClinical Tumor Immunology, Vol. 2, Kluwer Academic Publishers, Hingham,Mass.) and by Uhr J. of Immunol. 133:i-vii, 1984). In anotherillustrative example where the conjugate contains a radionuclidecytostatic agent, U.S. Pat. No. 4,897,255, Fritzberg et al.,incorporated herein by reference, is instructive of coupling methodsthat can be used to make the present conjugates.

The choice of coupling method will be influenced by the choice ofvascular smooth muscle binding protein or peptide, interstitial matrixbinding protein or peptide and therapeutic agent, and also by suchphysical properties as, e.g., shelf life stability, and/or by biologicalproperties, e.g., half-life in cells and blood, intracellularcompartmentalization route, and the like.

The physical and chemical character of the sustained release dosageforms of the present invention permit several alternative modes ofattachment of the dosage forms to binding proteins or peptides. Dosageforms (sustained release-type) of the present invention are capable ofbinding to binding proteins/peptides through, for example, covalentlinkages, intermediate ligand sandwich attachment, or non-covalentadsorption or partial entrapment. When the preferredpoly-lactic/glycolic acid particles are formed with the therapeuticagent dispersed therein, the uncharged polymer backbone is oriented bothinward (with the quasi lipophilic therapeutic agent contained therein)and outward, along with a majority of the terminal carboxy groups. Thesesurface carboxy groups may serve as covalent attachment sites whenactivated by, for example, a carbodiimide) for nucleophilic groups ofthe binding protein/peptide. Such nucleophilic groups include lysineepsilon-amino groups (amide linkage), serine hydroxyl groups (esterlinkage) or cysteine mercaptan groups (thioester linkage). Reactionswith particular groups depend upon pH and the reduction state of thereaction conditions.

For example, poly-lactic/glycolic acid particles having terminalcarboxylic acid groups can be reacted with N-hydroxybenztriazole in thepresence of a water soluble carbodiimide of the formula R—N═C═N—R′(wherein R is a 3-dimethylaminopropyl group or the like and R′ is anethyl group or the like). The benztriazole-derivatized particles (i.e.,activated imidate-bearing moieties) are then reacted with aprotein/peptide nucleophilic moiety such as an available epsilon-aminomoiety. Alternatively, p-nitrophenol, tetrafluorophenol,N-hydroxysuccinimide or like molecules are useful to form an activeester with the terminal carboxy groups of poly-lactic/glycolic acidparticles in the presence of carbodiimide. Other binding protein/peptidenucleophilic moieties include hydroxyl groups of serine, endogenous freethiols of cysteine, thiol groups resulting from reduction of bindingprotein/peptide disulfide bridges using reducing agents commonlyemployed for that purpose (e.g., cysteine, dithiothreitol,mercaptoethanol and the like) and the like. Additionally, the terminalcarboxy groups of the poly-lactic/glycolic acid particles areactivatable by reaction with thionyl chloride to form an acyl chloridederivatized moiety. The derivatized particles are then reacted withbinding peptide/protein nucleophilic groups to form targeted dosageforms of the present invention.

Direct conjugation of sustained release dosage form to binding proteinor peptide may disrupt binding protein/peptide recognition of the targetcell. Ligand sandwich attachment techniques are useful alternatives toachieve sustained release dosage form attachment to bindingprotein/peptide. These techniques involve the formation of a primarypeptide or protein shell using a protein that does not bind to thetarget cell population. Binding protein/peptide is then bound to theprimary peptide or protein shell to provide the resultant particle withfunctional binding protein/peptide. An exemplary ligand sandwichapproach involves covalent attachment of avidin or streptavidin to theparticles through functional groups as described above with respect tothe “direct” binding approach. The binding protein or peptide isderivatized, preferably minimally, via functionalized biotin (e.g.,through active ester, hydrazide, iodoacetal, maleimidyl or likefunctional groups). Ligand (i.e., binding peptide orprotein/functionalized biotin) attachment to the available biotinbinding sites of the avidin/streptavidin primary protein shell occursthrough the use of a saturating amount of biotinylated protein/peptide.

For example, poly-lactic/glycolic acid particles having terminalcarboxylic acid groups are activated with carbodiimide and subsequentlyreacted with avidin or streptavidin. The binding protein or peptide isreacted with biotinamidocaproate N-hydroxysuccinimide ester at a 1-3molar offering of biotin-containing compound to the bindingprotein/peptide to form a biotinylated binding protein/peptide. A molarexcess of the biotinylated binding protein/peptide is incubated with theavidin-derivatized particles to form a targeted dosage form of thepresent invention.

Alternatively, the particle carboxy groups are biotinylated (e.g.,through carbodiimide activation of the carboxy group and subsequentreaction with amino alkyl biotinamide). The biotinylated particles arethen incubated with a saturating concentration (i.e., a molar excess) ofavidin or streptavidin to form protein coated particles having freebiotin binding sites. These coated particles are then capable ofreaction with a molar excess of biotinylated binding protein formed asdescribed above. Another option involves avidin or streptavidin boundbinding peptide or protein attachment to biotinylated particles.

In addition, binding protein/peptide-particle attachment can be achievedby adsorption of the binding peptide to the particle, resulting from thenonionic character of the partially exposed polymer backbone of theparticle. Under high ionic strength conditions (e.g., 1.0 molar NaCl),hydrogen and hydrophobic particle-binding protein/peptide binding arefavored.

Moreover, binding protein/peptide may be partially entrapped in theparticle polymeric matrix upon formation thereof. Under thesecircumstances, such entrapped binding protein/peptide provides residualselective binding character to the particle. Mild particle formationconditions, e.g., those employed by Cohen et al., PharmaceuticalResearch, 8: 713-720 (1991), are preferred so as to entrap the proteinor peptide in the matrix. Entrapped binding proteins are also useful intarget cell reattachment of a partially degraded particle that hasundergone exocytosis. Binding proteins or peptides can be bound to otherpolymeric particle dosage forms (e.g., non-biodegradable dosage forms)having different exposed functional groups in accordance with theprinciples discussed above.

Exemplary non-biodegradable polymers useful in the practice of thepresent invention are polystyrenes, polypropylenes, styrene acrylic acidand acrylate copolymers and the like. Such non-biodegradable polymersincorporate or can be derivatized to incorporate functional groups forattachment of binding protein/peptide, including carboxylic acid groups,aliphatic primary amino groups, aromatic amino groups and hydroxylgroups.

Carboxylic acid functional groups are coupled to binding protein orpeptide using, for example, the reaction mechanisms set forth above forpoly-lactic/glycolic acid biodegradable polymeric particle dosage forms.Primary amino functional groups are coupled by, for example, reactionthereof with succinic anhydride to form a terminal carboxy moiety thatcan be bound to binding peptide/protein as described above.Additionally, primary amino groups can be activated with cyanogenbromide and form guanidine linkages with binding protein/peptide primaryamino groups. Aromatic amino functional groups are, for example,diazotized with nitrous acid to form diazonium moieties which react withbinding protein/peptide tyrosines, thereby producing a diazo bondbetween the non-biodegradable particle and the binding protein/peptide.Hydroxyl functional groups are coupled to binding protein/peptideprimary amino groups by, for example, converting the hydroxyl moiety toa moiety comprising a terminal carboxylic acid functional group. Thisconversion can be accomplished through reaction with chloroacetic acidfollowed by reaction with carbodiimide. Sandwich, adsorption andentrapment techniques, discussed above with respect to biodegradableparticles, are analogously applicable to non-biodegradableparticle-binding protein/peptide affixation.

In a preferred embodiment, targeting is specific for potentiallyproliferating cells that result in increased smooth muscle in theintimal region of a traumatized vascular site, e.g., followingangioplasty, e.g., pericytes and vascular smooth muscle cells. Aspectsof the invention relate to therapeutic modalities in which thetherapeutic conjugate of the invention is used to delay, reduce, oreliminate smooth muscle proliferation after angioplasty, e.g., PTCA,atheroectomy and percutaneous transluminal coronary rotationalatheroblation.

In another embodiment, targeting is specific for a local administrationaccessible pathologically proliferating or hyperactive normal cellpopulation implicated in, e.g., degenerative eye disease, cornealpannus, hyperactive endocrine glands or the like. Aspects of thisembodiment of the present invention involve therapeutic modalitieswherein the therapeutic agent reduces or eliminates proliferation orhyperactivity of the target c ell population.

Dosages, Formulation and Routes of Administration of the TherapeuticAgents

The amount of therapeutic agent administered is adjusted to treatvascular traumas of differing severity. For example, smaller doses aresufficient to treat lesser vascular trauma, e.g., to prevent vascularrejection following graft or transplant, while larger doses aresufficient to treat more extensive vascular trauma, e.g., to treatrestenosis following angioplasty. Thus, to biologically stent atraumatized vessel, a cytoskeletal inhibitor such as cytochalasin B isadministered at a systemic total dose of about 1 to about 24 ml,preferably about 1 to about 4 ml, at about 0.001 to about 25 μgcytochalasin/ml of vehicle, e.g., 0.01 to about 10 μg cytochalasin B/mlof vehicle, preferably about 0.1 to about 10 μg cytochalasin B/ml ofvehicle, and more preferably about 0.1 to about 8.0 μg cytochalasin B/mlof vehicle, although other dosages may prove beneficial. In particular,lower or higher concentrations of a cytochalasin may exert a therapeuticeffect when a non-aqueous solvent is employed as the vehicle.

The administration of a sytemic dose of cytochalasin B results in about5 to about 40, preferably about 8 to about 30, lambda of thecytochalasin B-containing solution entering the interstitial spacesurrounding the cells of the tunica media, and about 0.01 to about 4,preferably about 0.05 to about 3, ml of the solution being exposed tothe wall of the vessel by the transport of the solution to theadventitia. Moreover, these dosages may also exhibit anti-proliferativeeffects.

Administration of a therapeutic agent in accordance with the presentinvention may be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of the agents of theinvention may be essentially continuous over a preselected period oftime or may be in a series of spaced doses, e.g., either before, during,or after procedural vascular trauma, before and during, before andafter, during and after, or before, during and after the proceduralvascular trauma. Moreover, the administration of the therapeutic agentis selected so as to not further damage the traumatized vessel.

One or more suitable unit dosage forms comprising the therapeutic agentof the invention, which may be formulated for sustained release, can beadministered by a variety of routes including oral, or parenteral,including by rectal, transdermal, subcutaneous, intravenous,intramuscular, intrapulmonary and intranasal routes. When thetherapeutic agents of the invention are prepared for oraladministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, preferably in unit dosage form. The total activeingredients in these formulations can comprise from 0.1 to 99.9% byweight of the formulation. By “pharmaceutically acceptable” it is meantthe carrier, diluent, excipient, and/or salt must be compatible with theother ingredients of the formulation, and not deleterious to therecipient thereof.

Pharmaceutical formulations containing the therapeutic agent of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. For example, a cytochalasin canbe formulated with common excipients, diluents, or carriers, and formedinto tablets, capsules, suspensions, powders, and the like. Examples ofexcipients, diluents, and carriers that are suitable for suchformulations include the following fillers and extenders, e.g., starch,sugars, mannitol, and silicic derivatives; binding agents, for example,carboxymethyl cellulose, HPMC, and other cellulose derivatives,alginates, gelatin, and polyvinyl-pyrrolidone; moisturizing agents suchas glycerol; disintegrating agents, e.g., calcium carbonate and sodiumbicarbonate; agents for retarding dissolution, for example, paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents, e.g., cetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; and lubricants, for example,talc, calcium and magnesium stearate, and solid polyethyl glycols. SeeFondy et al. (WO 88/10259), the disclosure of which is incorporated byreference herein.

For example, tablets or caplets containing a therapeutic agent of theinvention can include buffering agents, e.g., calcium carbonate,magnesium oxide and magnesium carbonate. Caplets and tablets can alsoinclude inactive ingredients such as cellulose, pregelatinized starch,silicon dioxide, hydroxypropyl methylcellulose, magnesium stearate,microcrystalline cellulose, starch, talc, titanium dioxide, benzoicacid, citric acid, corn starch, mineral oil, polypropylene glycol,sodium phosphate, and zinc stearate, and the like. Hard or soft gelatincapsules containing a therapeutic agent of the invention can containinactive ingredients, for example, gelatin, microcrystalline cellulose,sodium lauryl sulfate, starch, talc, and titanium dioxide, and the like,as well as liquid vehicles such as polyethylene glycols (PEGs) andvegetable oil.

The therapeutic agents of the invention can also be formulated aselixirs or solutions for convenient oral administration or as solutionsappropriate for parenteral administration, for instance byintramuscular, subcutaneous or intravenous routes. In the practice ofcertain embodiments of the present invention, the therapeutic agent isdispersed in a pharmaceutically acceptable carrier that is in liquidphase, and delivered via an implantable device, e.g., a catheter. Usefulpharmaceutically acceptable carriers for these purposes includegenerally employed carriers, such as phosphate buffered saline solution,water, emulsions (e.g., oil/water and water/oil emulsions) and wettingagents of various types.

These formulations can contain pharmaceutically acceptable vehicles andadjuvants which are well known in the prior art. It is possible, forexample, to prepare solutions using one or more organic solvent(s) thatis/are acceptable from the physiological standpoint, chosen, in additionto water, from solvents, e.g., acetone, ethanol, isopropyl alcohol,glycol ethers such as the products sold under the name “Dowanol”,polyglycols and polyethylene glycols, C₁-C₄ alkyl esters of short-chainacids, preferably ethyl or isopropyl lactate, fatty acid triglycerides,for example products marketed under the name “Miglyol”, isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They can alsocontain gums, e.g., xanthan, guar or carbo gum or gum arabic, oralternatively polyethylene glycols, bentones and montmorillonites, andthe like.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, other preservatives, film-forming,keratolytic or comedolytic agents, perfumes and colorings. Also, otheractive ingredients may be added, whether for the conditions described orsome other condition.

For example, among antioxidants, t-butylhydroquinone, butylatedhydroxyanisole, butylated hydroxytoluene and α-tocopherol and itsderivatives may be mentioned. The galenical forms chiefly conditionedfor topical application take the form of creams, milks, gels,dispersions or microemulsions, lotions thickened to a greater or lesserextent, impregnated pads, ointments or sticks, or alternatively the formof aerosol formulations in spray or foam form or alternatively in theform of a cake of soap.

Additionally, the therapeutic agents are well suited to formulation assustained release dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or preferablyin a particular part of the intestinal tract, e.g., over a period oftime. The coatings, envelopes, and protective matrices may be made, forexample, from polymeric substances or waxes.

The local delivery of the therapeutic agents of the invention can be bya variety of techniques which administer the agent at or near thetraumatized vascular site. Examples of site-specific or targeted localdelivery techniques are not intended to be limiting but to beillustrative of the techniques available. Examples include localdelivery catheters, such as an infusion catheter, an indwellingcatheter, or a needle catheter, stets, synthetic grafts, adventitialwraps, shunts and stents or other implantable devices, site specificcarriers, direct injection, or direct applications.

Local delivery by an implant describes the surgical placement of amatrix that contains the therapeutic agent into the lesion ortraumatized area. The implanted matrix releases the therapeutic agent bydiffusion, chemical reaction, or solvent activators. See, for example,Lange, Science, 249, 1527 (1990).

An example of targeted local delivery by an implant is the use of astent. Stents are designed to mechanically prevent the collapse andreocclusion of the coronary arteries or other vessels. Incorporation ofa therapeutic agent into the stent can deliver the therapeutic agentdirectly to the lesion. Local delivery of agents by this technique isdescribed in Koh, Pharmaceutical Technology (October, 1990).

For example, a metallic, plastic or biodegradable intravascular stent isemployed which comprises the therapeutic agent. The stent preferablycomprises a biodegradable coating, a porous or a permeablenon-biodegradable coating, or a biodegradable or non-biodegradablemembrane or synthetic graft sheath-like coating, e.g., PTFE, comprisingthe therapeutic agent. A more preferred embodiment of the invention is acoated stent wherein the coating comprises a sustained-release dosageform of the therapeutic agent. In an alternative embodiment, abiodegradable stent may also have the therapeutic agent impregnatedtherein, i.e., in the stent matrix.

A biodegradable stent with the therapeutic agent impregnated therein canbe further coated with a biodegradable coating or with a porousnon-biodegradable coating having the sustained release-dosage form ofthe therapeutic agent dispersed therein. This stent can provide adifferential release rate of the therapeutic agent, i.e., there can bean initial faster release rate of the therapeutic agent from thecoating, followed by delayed release of the therapeutic agentimpregnated in the stent matrix, upon degradation of the stent matrix.The intravascular stent also provides a mechanical means of providing anincrease in luminal area of a vessel.

Furthermore, the placement of intravascular stents comprising atherapeutic agent which is an inhibitor of smooth muscle cellproliferation can also reduce or prevent intimal proliferation. Thisinhibition of intimal smooth muscle cells and stroma produced by thesmooth muscle and pericytes can lead to a more rapid and completere-endothelization following the intraventional placement of thevascular stent. The increased rate of re-endothelization andstabilization of the vessel wall following stent placement can reducethe loss of luminal area and decreased blood flow due to vascular smoothmuscle cell proliferation which is one of the primary causes of vascularstent failures.

Another example of targeted local delivery by an implant is the use ofan adventitial wrap. The wrap comprises a pharmaceutically acceptablecarrier matrix, e.g., a Pluronic gel which is free, or contained by acollagen mesh, which gel has dispersed therein a therapeutic agent. Oneembodiment of the invention is a pluronic gel (F-127, BASF) which issoluble at 4° C. but solidifies at 37° C., e.g., on contact with fluidor tissue in a human. To prepare a pluronic gel containing wrap, 4 ml ofphosphate buffer, pH 7.0 (Circ. Res., vol. 76, April 1995), was added to1 g of pluronic gel F-127, which was mixed overnight at 4° C. Thetherapeutic agent was added to the mixture prior to localadministration. The mixture may be applied directly to a surgicallyexposed artery wall, or may be applied to the surface of a bovinecollagen mat (BioCore, Inc., Topeka, Kans.), which is then wrappedaround the artery and the edges joined by sutures.

Another embodiment of the invention is the incorporation of thetherapeutic agent into the expanded nodal spaces of a PTFE (Impra, Inc.,Tempe, Ariz.) vascular graft-like membrane which can surround, or beplaced on the interior or on the exterior surface of, an interlumenalvascular stent, which comprises metal or a biodegradable ornonbiodegradable polymer. The therapeutic agent, or a sustained releasedosage form of the therapeutic agent, fills the nodal spaces of the PTFEmembrane wall and/or coats the inner and/or outer surfaces of themembrane.

Yet another embodiment of the invention is a mixture of a crystallineform of a therapeutic agent in a bovine collagen gel (BioCore, Inc.,Topeka, Kans.). Crystals varied in size from about 0.1 micron to about 1mm. Generally, the crystals were pulverized to generate smaller sizedcrystals. This mixture is applied directly to the surface of the artery,and the surrounding subcutaneous tissues sutured around the vessel andthe skin closed. Bovine collagen (BioCore, Inc., Topeka, Kans.) isdissolved in sterile saline (1:1) and the crystalline therapeutic agentadded. Alternatively, the collagen gel is applied to a bovine collagenmesh which is then wrapped around the vessel and the edges sutured tohold the mesh in place. The bovine collagen mesh (BioCore, Inc.) is cutto size, e.g., 1 cm×1 cm, and the therapeutic agent-collagen gel mixtureis applied to the surface of the mesh.

A further embodiment of the invention comprises the entrapment ofcrystalline therapeutic agent in about a 0.1 to about 3, preferablyabout 0.5 to about 0.7, mm thick silicone membrane, e.g., siliconepolymer Q-7 4840 (Dow Corning, Midland, Mich.). The polymer (part A andB, 1:1) is mixed with a spatula. An inert filler, e.g., mannitol, ispowdered and sieved to a fraction 53-75 mesh size. Mannitol andtherapeutic agent are mixed in predetermined proportions and thenlevigated with the polymer to form a composite. The composite is filledin a slab mold and compressed to 5000 psi. The composite is then curedat 80° C. for 2 hours. The composite membrane is then cut to size, e.g.,1 cm×1 cm, wrapped around the artery and held in place by suturing themembrane edges together.

A therapeutic agent may also be coated onto the exterior of the wrap.The wrap and/or the coating is preferably biodegradable. It is preferredthat the therapeutic agent is in sustained release dosage form.

Another example is a delivery system in which a polymer that containsthe therapeutic agent is injected into the area of the lesion in liquidform. The polymer then solidifies or cures to form the implant which isretained in situ. This technique is described in PCT WO 90/03768 (Donn,Apr. 19, 1990).

Another example is the delivery of a therapeutic agent by polymericendoluminal sealing. This technique uses a catheter to apply a polymericimplant to the interior surface of the lumen. The therapeutic agentincorporated into the biodegradable polymer implant and is therebyreleased at the surgical site. This technique is described in PCT WO90/01969 (Schindler, Aug. 23, 1989), the disclosure of which isincorporated by reference herein.

Yet another example of local delivery is by direct injection of vesiclesor microparticles into the lesion or artery wall adjacent to the lesion.These microparticles may be composed of substances such as proteins,lipids, carbohydrates or synthetic polymers. These microparticles havethe therapeutic agent incorporated throughout the microparticle or ontothe microparticle as a coating. Delivery systems incorporatingmicroparticles are described in Lange, Science, 249,1527 (1990) andMathiowitz et al., J. App. Poly. Sci., 26, 809 (1981).

For topical administration, the therapeutic agents may be formulated asis known in the art for direct application to a target area.Conventional forms for this purpose include wound dressings, coatedbandages or other polymer coverings, ointments, lotions, pastes,jellies, sprays, and aerosols. The percent by weight of a therapeuticagent of the invention present in a topical formulation will depend onvarious factors, but generally will be from 0.005% to 95% of the totalweight of the formulation, and typically 1-25% by weight.

Conditions Amenable to Treatment by the Method of the Invention

The therapeutic agents and dosage forms of the invention are useful totreat or inhibit a diminution in vessel lumen volume, area and/ordiameter associated with a procedural vascular trauma. A vascular traumaincludes but is not limited to trauma associated with an interventionalprocedure, such as angioplasty, placement of a stent, shunt, stet,synthetic or natural graft, adventitial wrap, indwelling catheter orother implantable devices. Grafts include synthetic therapeuticagent-treated grafts, e.g., impregnated or coated grafts. As usedherein, “vessels” includes mammalian vessels, e.g., coronary vessels aswell as peripheral, femoral and carotid vessels. It will be recognizedthat the therapeutic agents and dosage forms (both free and sustainedrelease) of the invention are not restricted in use for therapyfollowing angioplasty; rather, the usefulness of the therapeutic agentsand dosage forms will be proscribed by their ability to inhibit cellularactivities of smooth muscle cells and pericytes in the vascular wall.Thus, other aspects of the invention include therapeutic conjugates anddosage forms and protocols useful in early therapeutic intervention forreducing, delaying, or eliminating (and even reversing) atheroscleroticplaques and areas of vascular wall hypertrophy and/or hyperplasia.

The therapeutic agents and dosage forms of the invention are also usefulin therapeutic modalities for enhancing the regrowth of endothelialcells in injured vascular tissues and in other wound sites includingepithelial wounds. In these therapeutic modalities, the therapeuticagents, conjugates and dosage forms of the invention find utility ininhibiting the migration and/or proliferation of smooth muscle cells orpericytes. Smooth muscle cells and pericytes have been implicated in theproduction of factors in vitro that inhibit endothelial cellproliferation, and their proliferation can also result in a physicalbarrier to establishing a continuous endothelium. Thus, the therapeuticagents, conjugates and dosage forms of the invention find utility inpromoting neo-angiogenesis and increased re-endothelialization, e.g.,during wound healing, vessel grafts and following vascular surgery. Thedosage forms may also release therapeutic modalities that stimulate oraccelerate up re-endothelialization of the damaged vessel wall. Anexemplary therapeutic agent for this purpose is vascular permeabilityfactor.

One embodiment of the present invention involves administration of atherapeutic agent capable of inhibiting the ability of vascular smoothmuscle cells to contract and/or proliferate and/or migrate. Exemplaryagents useful in the practice of this aspect of the present inventionare those capable of causing a traumatized artery to lose vascular tone,such that normal vascular hydrostatic pressure (i.e., blood pressure)expands the flaccid vessel to or near to its maximal physiologicaldiameter. Loss of vascular tone may be caused by agents that interferewith the formation or function of contractile proteins (e.g., actin,myosin, tropomyosin, caldesmon, calponin or the like). This interferencecan occur directly or indirectly through, for example, inhibition ofcalcium modulation, phosphorylation or other metabolic pathwaysimplicated in contraction of vascular smooth muscle cells.

Inhibition of cellular contraction (i.e., loss of vascular tone) mayoperate through two mechanisms to reduce the degree of vascularstenosis. First, inhibition of cellular contraction for a prolongedperiod of time limits the number of smooth muscle cells that migratefrom the tunica media into the intima, the thickening of which resultsin vascular luminal stenosis. Second, inhibition of cellular contractioncauses the smooth muscle wall to relax and dilate under normal vascularhydrostatic pressure (i.e., blood pressure). Therapeutic agents, e.g.,the cytochalasins, inhibit smooth muscle cell contraction withoutabolishing the protein synthesis necessary for traumatized,post-angioplasty or other surgically- or disease-damaged, smooth musclecells to repair themselves. Protein synthesis is also necessary for thesmooth muscle cells to secrete matrix, which fixes or retains the lumenin a state near its maximum systolic diameter as the vascular lesionstabilizes (i.e., a biologically-induced stenting effect).

This biological stenting effect not only results in an expanded vesselluminal cross-sectional area or diameter and increased blood flow ratethrough the vessel, but also significantly reduces elastic recoilfollowing angioplasty. Elastic recoil is an acute closure of the vesselassociated with vasospasm or early relaxation of the muscular wall, dueto trauma shock resulting from vessel over-stretching by a ballooncatheter during angioplasty. This spasm of the tunica media which leadsto decreases in the luminal cross-sectional area may occur within hours,days or weeks after the balloon dilation, as restoration of vascularmuscle wall tone occurs.

Recent observations during microscopic examination of atheroectomyspecimens suggest that elastic recoil may occur in up to 25% ofangioplasty procedures classified as successful, based on the initialpost-procedure angiogram. Because the biological stenting procedurerelaxes the artery wall following balloon angioplasty, the clinician caneliminate over-inflation and its resultant trauma shock as a means todiminish or delay the vessel spasm or elastic recoil. Reduction orelimination of over-inflation decreases trauma to the muscular wall ofthe vessel, thereby reducing the determinants of smooth muscle cellproliferation in the intima and, therefore, reducing the incidence orseverity of restenosis.

Biological stenting also decreases the incidence of thrombus formation.In pig femoral arteries treated with cytochalasin B, for example, theincidence of mural microthrombi was decreased as compared to the balloontraumatized arteries that were not treated with the therapeutic agent.This phenomenon appears to be a secondary benefit that may result fromthe increased blood flow through the traumatized vessel, said benefitbeing obtained through the practice of the present invention. Inarteries treated with sustained release dosage forms of cytochalasin B,cytochalasin B may also prevent the contraction and organization ofplatelets which is required for thrombus formation.

Cytochalasins are exemplary therapeutic agents capable of generating abiological stenting effect on vascular smooth muscle cells.Cytochalasins are thought to inhibit both migration and contraction ofvascular smooth muscle cells by interacting with actin. Thecytochalasins interact with the ends of filamentous actin to inhibit theelongation of the actin filaments. Low doses of cytochalasins (e.g.,cytochalasin B) also disrupt microfilament networks of actin. In vitrodata indicate that after vascular smooth muscle cells clear cytochalasinB, the cells regenerate enough polymerized actin to resume migrationwithin about 24 hours. In vivo assessments reveal that vascular smoothmuscle cells regain vascular tone within 2 to 4 days. It is during thisrecuperative period that the lumen diameter fixation and biologicalstenting effect occurs.

The therapeutic agent may be targeted, but is preferably administereddirectly to the traumatized vessel prior to, during or following theangioplasty or other traumatic event. The biological stenting effect ofcytochalasin B, for example, is achievable using a single infusion ofabout 1 to about 24 ml, preferably about 5 to about 15 ml, of a vehicleplus the therapeutic agent into the traumatized region of the vesselwall at a dose concentration ranging from about 0.1 microgram oftherapeutic agent/ml of vehicle to about 1.0 microgram, preferably aboutabout 0.1 microgram of therapeutic agent/ml of vehicle to about 10.0microgram, and more preferably about about 0.01 microgram of therapeuticagent/ml of vehicle to about 10.0 microgram, of therapeutic agent/ml ofvehicle.

Inhibition of vascular smooth muscle cell migration (from the tunicamedia to the intima) has been demonstrated in the same dose range(Example 11); however, a sustained exposure of the vessel to thetherapeutic agent is preferable in order to maximize theseanti-migratory effects. If the vascular smooth muscle cells cannotmigrate into the intima, they cannot proliferate there. Should vascularsmooth muscle cells migrate to the intima, a subsequently administeredanti-proliferative sustained release dosage form can inhibit intimalproliferation. As a result, the sustained release dosage form of thepresent invention, incorporating a cytochalasin or otheranti-proliferative therapeutic agent, can be administered in combinationwith a free therapeutic agent which is preferably a cytoskeletalinhibitor. In this manner, a biological stenting effect, as well as ananti-proliferative or anti-migratory effect, can be achieved in a singledosing protocol.

The present invention also provides methods for the treatment of cancerand immune system-mediated diseases through local administration of atargeted particulate dosage form. The particulate dosage form is, forexample, administered locally into primary and/or metastatic foci ofcancerous target cells. Local administration is preferably conductedusing an infusion needle or intraluminal administration route, infusingthe particulate dosage form in the intercellular region of the tumortissue or in luminal fluid surrounding the tumor cells.

Primary foci introduction is preferably conducted with respect to targetcells that are generally situated in confined areas within a mammal,e.g., ovarian carcinomas located in the abdominal cavity. The dosageform of the present invention binds to the target cell population and,optionally, is internalized therein for release of the therapeutic agentover time. Local administration of dosage forms of the present inventionto such primary foci results in a localized effect on such target cells,with limited exposure of other sensitive organs, e.g., the bone marrowand kidneys, to the therapeutic agent.

When metastatic foci constitute the target cell population, theadministered microparticles and larger nanoparticles are primarily boundto the target cells situated near the infusion site and are, optionally,internalized for release of the therapeutic agent, thereby generating amarked and localized effect on the target cells immediately surroundingthe infusion site. In addition, smaller nanoparticles followinterstitial fluid flow or lymphatic drainage channels and bind totarget cells that are distal to the infusion site and undergoinglymphatic metastasis.

The targeted dosage forms of this embodiment of the present inventioncan be used in combination with more commonly employed immunoconjugatetherapy. In this manner, the immunoconjugate achieves a systemic effectwithin the limits of systemic toxicity, while the dosage form of thepresent invention delivers a concentrated and sustained dose oftherapeutic agent to the primary and metastatic foci, which oftenreceive an inadequate therapeutic dose from such “systemic”immunoconjugate administration alone, and avoids or minimizes systemictoxic effects.

Where the target cell population can be accessed by localadministration, the dosage forms of the present invention are utilizedto control immune system-mediated diseases. Exemplary of such diseasesare arthritis, sprue, uveitis, endophthalmitis, keratitis and the like.The target cell populations implicated in these embodiments of thepresent invention are confined to a body cavity or space, such as jointcapsules, pleural and abdominal cavity, eye and sub-conjunctival space,respectively. Local administration is preferably conducted using aninfusion needle for a intrapleural, intraperitoneal, intraocular orsub-conjunctival administration route.

This embodiment of the present invention provides a more intense,localized modulation of immune system cells with minimal effect on thesystemic immune system cells. Optionally, the systemic cells of theimmune system are simultaneously treatable with a chemotherapeutic agentconjugated to a binding protein or peptide. Such a conjugate preferablypenetrates from the vascular lumen into target immune system cells.

The local particulate dosage form administration may also localize tonormal tissues that have been stimulated to proliferate, therebyreducing or eliminating such pathological (i.e., hyperactive)conditions. An example of this embodiment of the present inventioninvolves intraocular administration of a particulate dosage form coatedwith a binding protein or peptide that localizes to pericytes and smoothmuscle cells of neovascularizing tissue. Proliferation of thesepericytes causes degenerative eye disease. Preferred dosage forms of thepresent invention release compounds capable of suppressing thepathological proliferation of the target cell population. The preferreddosage forms can also release compounds that increase vessel lumen areaand blood flow, reducing the pathological alterations produced by thisreduced blood supply.

Method of the Invention

The invention provides a method of treating a mammal having, or at riskof, diminution in vessel lumen volume, area or diameter, e.g., stenosisor restenosis of a blood vessel. The method comprises the administrationof at least one therapeutic agent in an amount effective to biologicallystent a vessel, inhibit or reduce vascular remodeling of a vessel,inhibit or reduce vascular smooth muscle cell proliferation, or anycombination thereof.

For the prevention of vessel lumen diminution associated with proceduralvascular trauma, the therapeutic agent can be administered before,during and/or after the procedure, or any combination thereof. Forexample, for the prevention of restenosis, a series of spaced doses ofthe therapeutic agent, optionally, in sustained release dosage form, ispreferably administered before, during and/or after the traumaticprocedure (e.g., angioplasty). The dose may also be delivered locally,via an implantable device, e.g., a catheter, introduced into theafflicted vessel during the procedure. Preferably, a sustained releasedosage form is administered via the implantable device during thetraumatic procedure. After the traumatic procedure is conducted, aseries of follow-up doses can be administered over time, preferably in asustained release dosage form, for a time sufficient to substantiallyreduce the risk of, or to prevent, restenosis. A preferred therapeuticprotocol duration for this purpose involves administration from about 3to about 26 weeks after angioplasty.

It will be recognized by those skilled in the art thattherapeutically/prophylactically effective dosages of the therapeuticagents and dosage forms will be dependent on several factors, including,e.g.: a) the binding affinity of the vascular smooth muscle bindingprotein, if any, in the dosage form; b) the atmospheric pressure andduration of the pressure applied during infusion; c) the time over whichthe therapeutic agent or dosage form administered resides at thevascular site; d) the nature of the therapeutic agent employed; e) thenature of the vascular trauma and therapy desired; f) for sustainedrelease dosage forms, the rate of release of the therapeutic agent fromthe dosage form, and/or g) for sustained release dosage forms, theintercellular and/or intracellular localization of the dosage form.Those skilled practitioners trained to deliver drugs at therapeuticallyeffective dosages (e.g., by monitoring drug levels and observingclinical effects in patients) will determine the optimal dosage for anindividual patient based on experience and professional judgment. Thoseskilled in the art will recognize that infiltration of the therapeuticagent into the intimal layers of a traumatized vessel wall in free orsustained release dosage form is subject to variation and will need tobe determined on an individual basis.

A therapeutically effective dosage of the therapeutic agent will betypically reached when the concentration of therapeutic agent in thefluid space between the balloons of the catheter is in the range ofabout 10⁻¹ to 10⁻¹² M. It will be recognized from the Examples providedherewith that therapeutic agents and dosage forms of the invention mayonly need to be delivered in an anti-proliferative therapeutic dosagesufficient to expose the proximal 6 to 9 cell layers of the intimal ortunica media cells lining the lumen to the therapeutic agent, whereasthe anti-contractile dosage may need to expose the entire tunica media.Alternatively, the anti-proliferative therapeutic dosage sufficient toexpose the inner 10%, preferably the inner 20%, and more preferably theinner 99%, of the tunica media cells lining the lumen to the therapeuticagent. This dosage can be determined empirically, e.g., by a) infusingvessels from suitable animal model systems and usingimmunohistochemical, fluorescent or electron microscopy methods todetect the agent and its effects (e.g., such as exemplified in theEXAMPLES below); and b) conducting suitable in vitro studies (see, forexample, EXAMPLES 3, 4, and 5, below).

For example, with respect to catheter delivery, it will be recognized bythose skilled in the art that therapeutically/prophylactically effectivedosages of the therapeutic agents and dosage forms will be dependent onfactors including: a) the atmospheric pressure applied during infusion;b) the time over which the agent administered resides at the vascularsite; c) the form of the therapeutic or prophylactic agent employed;and/or d) the nature of the vascular trauma and therapy desired.Catheters which may be useful in the practice of the invention includecatheters such as those disclosed in Just et al. (U.S. Pat. No.5,232,444), Abusio et al. (U.S. Pat. No. 5,213,576), Shapland et al.(U.S. Pat. No. 5,282,785), Racchini et al. (U.S. Pat. No. 5,458,568),Wolinsky (U.S. Pat. No. 4,824,436), Spears (U.S. Pat. No. 4,512,762) andShaffer et al. (U.S. Pat. No. 5,049,132), the disclosures of which areincorporated by reference herein.

It will be recognized that where the therapeutic conjugate or dosageform is to be delivered with an infusion catheter, the therapeuticdosage required to achieve the desired inhibitory activity for atherapeutic conjugate or dosage form can also be determined through theuse of in vitro studies. In a preferred aspect, the infusion cathetermay be conveniently a double balloon or quadruple balloon catheter witha permeable membrane. In one representative embodiment, atherapeutically effective dosage of a therapeutic conjugate or dosageform is useful in treating vascular trauma resulting from disease (e.g.,atherosclerosis, aneurysm, or the like) or vascular surgical proceduressuch as angioplasty, atheroectomy, placement of a stent (e.g., in avessel), thrombectomy, and grafting. Atheroectomy may be performed, forexample, by surgical excision, ultrasound or laser treatment, or by highpressure fluid flow. Grafting may be, for example, vascular graftingusing natural or synthetic materials or surgical anastomosis of vesselssuch as, e.g., during organ grafting. Those skilled in the art willrecognize that the appropriate therapeutic dosage for a given vascularsurgical procedure (above) is determined in in vitro and in vivo animalmodel studies, and in human preclinical trials.

Thus, in a preferred embodiment, about 0.3 atm (i.e., 300 mm of Hg) toabout 3 atm of pressure applied for 15 seconds to 3 minutes to thearterial wall is adequate to achieve infiltration of a sustained releasedosage form bound to the NR-AN-01 binding protein into the smooth musclelayers of a mammalian artery wall. Wolinsky et al., “DirectIntraarterial Wall Injection of Microparticles Via a Catheter: APotential Drug Delivery Strategy Following Angioplasty,” Am. HeartJour., 122(4):1136-1140, 1991. Those skilled in the art will recognizethat infiltration of a sustained release dosage form into a target cellpopulation will probably be variable and will need to be determined onan individual basis.

A therapeutically effective dosage is generally the pericellular agentdosage in smooth muscle cell tissue culture, i.e., a dosage at which ata continuous exposure results in a therapeutic effect between the toxicand minimal effective doses. This therapeutic level is obtained in vivoby determining the size, number and therapeutic agent concentration andrelease rate required for particles infused between the smooth musclecells of the artery wall to maintain this pericellular therapeuticdosage. The dosage form should release the therapeutic agent at a ratethat approximates the pericellular dose of the following exemplarytherapeutic agents: from about 0.01 to about 100 micrograms/mlnitroglycerin, from about 1.0 to about 1000 micrograms/ml of suramin,from about 0.001 to about 100 micrograms/ml for cytochalasin, and fromabout 0.01 to about 10⁵ nanograms/ml of staurosporin as well as fromabout 0.001 to about 100 micrograms/ml taxol. Thus, for cytochalasin B,the sytemic dose results in about 5 to about 40, preferably about 8 toabout 30, lambda of the solution entering the interstitial spacesurrounding the cells of the tunica media, and about 0.01 to about 4,preferably about 0.05 to about 3, ml of the solution delivered to thewall of the vessel via the adventitia.

The administration of a cellular therapeutic dose of, for example,cytochalasin B to vascular smooth muscle cells following balloondilation trauma can be achieved by replacing the entire volume of thetunica media with the therapeutic agent so as to produce a biostentingeffect, i.e., all cells are exposed to a concentration of thetherapeutic agent effective to biologically stent the vessel. Forexample, a dose response study which employed swine femoral arteriesshowed that if the entire tunica media was infused using an infusioncatheter with cytochalasin B in a range of about 0.1 μg/ml of vehicle to10.0 μg/ml of vehicle, a biostenting effect resulted. That is, a moreextensive retention of the artery lumen size (diameter orcross-sectional area) was observed relative to the artery lumen sizeproduced by the dilating balloon. The therapeutic effect had a thresholdlevel of 0.1 μg/ml cytochalasin B with no effect below this dose and noincrease in therapeutic efficacy up to 10 μg/ml cytochalasin B. Thus,cytochalasin B has a wide therapeutic index which ranges from about 0.1to about 10 μg/ml, with no evidence of toxicity at 10 μg/ml. Ten μg/mlis the maximum saturation concentration of cytochalasin B in saline. Thetherapeutic effect produced by cytochalasin B administration became moreapparent over the 3 to 8 weeks following the balloon trauma.

To achieve a cellular therapeutic dose to produce a biostenting effect,i.e., one where each cell of the tunica media is exposed to atherapeutic concentration range, e.g., about 0.1 μg/ml to about 8.0μg/ml of cytochalasin B, a catheter, e.g., a MIC2 or MIC3 catheter, isfilled with a volume of the therapeutic agent in solution and deliveredat a hub pressure which does not damage the vessel. For example, 9 to 24ml (MIC2) or 5 to 10 ml (MIC3) of an 8.0 μg/ml cytochalasin B solutionis delivered at a hub pressure of 4 to 5 atmospheres for a total of 90seconds infusion time. Delivery of the NR-ML-05 monoclonal antibody tothe tunica media under these conditions was achieved in both the swinefemoral and coronary models. With the hub pressure and exposure timesheld constant, the amount of solution infused may vary because flow rateis determined by the tightness of fit. If the flow rate is below therecommended range, the fit is too tight to establish a uniformhydrostatic head, and therefore a uniform dose around the vessel walldoes not exist. If the flow rate is above the recommended range, thenthe fit is too loose to establish the hydrostatic head required to forcethe solution into the interstitial region of the tunica media, and thedose to the individual cells is below the required therapeutic level.

It is also preferred that catheter administration of the therapeuticagent to the tunica media and adventitia produces minimal or no damageto the vessel wall by jetting or accentuating dissections produced bythe PTCA procedure. Preferably, about 1 to about 1.5 mls are infusedinto 1 to about 3 traumatized lesion sites. Also preferably, theinfusion is accomplished at a pressure of about 0.3 to about 5 atm forabout 15 seconds to about 3 minutes. It is preferred that thehydrostatic head pressure at the interface of the infusion balloon andthe vessel wall is between about 0.3 to about 1.5, preferably about 0.4to about 0.8, and more preferably about 0.5 to about 0.75, atmospheres,so as to rapidly force the solution containing the therapeutic agentinto the tunica media interstitial space without rupturing the smallvessels in the adventitia, the origin of which is exposed to thehydrostatic head. Preferably, the infusion is accomplished by a hubpressure of about 4 to about 5 atm for a period of time from about 1 toabout 4 minutes, preferably about 1 to 2 minutes. This infusion regimewill result in the penetration of an efficacious dose of the therapeuticagent to the smooth muscle cells of the vessel wall. Preferably, thetherapeutic agent will be at a concentration of from about 0.01 μg/ml toabout 8.0 μg/ml of infusate. Preferably, the therapeutic agent is acytochalasin, and more preferably, cytochalasin B, or a functionallyequivalent analog thereof.

Preferably, the administration of the therapeutic agent results inuniform delivery of the therapeutic agent to the tunica media. Moreover,the catheter administration of the therapeutic agent results in thedelivery of an effective amount of the agent to the adventitia via thevasa vasorum, as well as the tunica media. Preferably, the therapeuticagent is also uniformly distributed to the adventitia. Catheteradministration of a therapeutic agent, e.g., about 4 to about 24 ml ofcytochalasin B at about 8.0 μg/ml, preferably results in a uniformpattern of therapeutic agent delivery, at a depth of penetration to atleast about the inner 10%, more preferably to at least about the inner20%, even more preferably to the inner 100%, of the tunica media. By thetime a therapeutic agent diffused from the inner 10% of the tunica mediato its outer limits in a swine coronary study, it was found that thecells at the outer limits were exposed to a therapeutic dose.

A preferred form of the therapeutic agent includes cytochalasin B insterile saline at a concentration of about 8.0 μg/ml, in 30 ml vials,which should deliver the preferred cellular therapeutic dose describedhereinabove needed to treat a single lesion. Preferably, the amount isuniformly distributed to the inner 20% of the tunica media and uniformlydistributed to the adventitia.

In another embodiment of the present invention, a solution of atherapeutic agent is infused, in vivo or ex vivo, into the walls ofisolated vessels (arteries or veins) to be used for vascular grafts. Inthis embodiment of the invention, the vessel that is to serve as thegraft is excised or isolated and subsequently distended by an infusionof a solution of a therapeutic agent. Preferably the infusion isaccomplished by a hub pressure of about 4 to about 5 atm for a period oftime from about 1 to about 4 minutes, preferably about 1 to 2 minutes.This infusion regime will result in the penetration of an efficaciousdose of the therapeutic agent to the smooth muscle cells of the vessel.Wall. Preferably, the therapeutic agent will be at a concentration offrom about 0.01 μg/ml to about 0.8 μg/ml, more preferably from about0.01 μg/ml to about 8.0 μg/ml of infusate. Preferably, the therapeuticagent is a cytochalasin, and more preferably, cytochalasin B, or afunctionally equivalent analog thereof.

It is known to those of ordinary skill in the art that peripheralvessels that are used for vascular grafts in other peripheral sites orin coronary artery bypass grafts, frequently fail due to post surgicalstenosis. Since cytochalasin B infusion maintains the vascular luminalarea in surgically traumatized vessels by virtue of its biologicalstenting activity, its administration in this process can retard theability of the vessel to contract, resulting in a larger luminaldiameter or cross-sectional area. Furthermore, it is an advantage ofthis embodiment of the present invention that the administration ofcytochalasin B in this manner can prevent the constriction or spasm thatfrequently occurs after vascular grafts are anastomosed to both theirproximal and distal locations, that can lead to impaired function, ifnot total failure, of vascular grafts. Thus, the vessel stentingproduced by cytochalasins should decrease the incidence of spasms, whichcan occur from a few days to several months following the graftprocedure.

For example, in another embodiment of the invention, the therapeuticagents and dosage forms may be used in situations in which angioplastyis not sufficient to open a blocked artery, such as those situationswhich require the insertion of an intravascular stent or shunt or otherimplantable devices. Thus, the invention also provides stents, stets,adventitial wraps, indwelling catheters, synthetic grafts or shuntscomprising the therapeutic agent. Useful therapeutic agents in thisembodiment of the invention include anti-proliferative agents, e.g.,cytoskeletal inhibitors. A preferred cytoskeletal inhibitor is acytochalasin, for example, cytochalasin B or an analog thereof which isa functional equivalent of cytochalasin B. Another preferredcytoskeletal inhibitor of the invention is taxol or an analog of taxolwhich is a functional equivalent of taxol. Preferably, theani-proliferative agent is in sustained release dosage form.

Thus, an implantable device, e.g., an intravascular stent or shunt,provides a mechanical means of providing an increase in luminal diameterof a vessel, in addition to that provided via the biological stentingaction and/or anti-proliferative activity of the cytoskeletal inhibitor,such as cytochalasin B or taxol, releasably embedded therein oradministered in solution or suspension during the interventionalprocedure. Furthermore, the placement of an implantable devicecomprising a therapeutic agent which is an inhibitor of smooth musclecell proliferation provides an increased efficacy by reducing orpreventing intimal proliferation. This inhibition of intimal smoothmuscle cells and stroma produced by the smooth muscle allows for morerapid and complete re-endothelization following the intraventionalplacement of the device.

For the therapeutic conjugates of the invention, non-coupled vascularsmooth muscle cell binding protein (e.g., free NR-AN-01 antibody) ispreferably administered prior to administration of the therapeutic agentconjugate or dosage form to provide a blocker of non-specific binding tocross-reactive sites. Blocking of such sites is important becausevascular smooth muscle cell binding proteins will generally have somelow level of cross-reactivity with cells in tissues other than thedesired smooth muscle cells. Such blocking can improve localization ofthe therapeutic conjugate or dosage form at the specific vascular site,e.g., by making more of the therapeutic conjugate available to thecells. As an example, non-coupled vascular smooth muscle binding proteinis administered from about 5 minutes to about 48 hours, most preferablyfrom about 5 minutes to about 30 minutes, prior to administration of thetherapeutic conjugate or dosage form. In one preferred embodiment of theinvention, the unlabeled specific “blocker” is a monovalent or bivalentform of an antibody (e.g., a whole antibody or an F(ab)′₂, Fab, Fab′, orFv fragment of an antibody). The monovalent form of the antibody has theadvantage of minimizing displacement of the therapeutic conjugate ordosage form while maximizing blocking of the non-specific cross-reactivesites. The non-coupled vascular smooth muscle cell binding protein isadministered in an amount effective to blocking binding of a least aportion of the non-specific cross-reactive sites in a patient. Theamount may vary according to such factors as the weight of the patientand the nature of the binding protein. In general, about 0.06 mg to 0.20mg per kg body weight or more of the unlabeled specific blocker isadministered to a human.

In addition, a second irrelevant vascular smooth muscle cell bindingprotein may optionally be administered to a patient prior toadministration of the therapeutic conjugate or dosage form to reducenon-specific binding of the therapeutic conjugate or dosage form totissues. In a preferred embodiment, the irrelevant binding protein maybe an antibody which does not bind to sites in the patient throughantigen-specific binding, but instead binds in a non-specific manner,e.g., through Fc receptor binding reticuloendothelial cells,asialo-receptor binding, and by binding to ubiquitin-expressing cells.The irrelevant “blocker” decreases non-specific binding of thetherapeutic conjugate or dosage form and thus reduces side-effects,e.g., tissue toxicity, associated with the use of the therapeuticconjugate or dosage form. The irrelevant “blocker” is advantageouslyadministered from 5 minutes to 48 hours, most preferably from 15 minutesto one hour, prior to administration of the therapeutic conjugate ordosage form, although the length of time may vary depending upon thetherapeutic conjugate and route or method of injection. Representativeexamples of irrelevant “blockers” include antibodies that arenonreactive with human tissues and receptors or cellular and serumproteins prepared from animal sources that when tested are found not tobind in a specific manner (e.g., with a Ka<10³ M⁻¹) to human cellmembrane targets.

Kits Comprising an Implantable Delivery Device and At Least OneTherapeutic Agent of the Invention

The invention provides a kit comprising packing material enclosing,separately packaged, at least one implantable device adapted for thedelivery of a therapeutic agent, e.g., a catheter, an adventitial wrap,a valve, a stent, a stet, a shunt or a synthetic graft, and at least oneunit dosage form comprising the therapeutic agent, as well asinstruction means for their use, in accord with the present methods. Theunit dosage form may comprise an amount of at least one of the presenttherapeutic agents effective to accomplish the therapeutic resultsdescribed herein when delivered locally and/or systemically. A preferredembodiment of the invention is a kit comprising a catheter adapted forthe local delivery of at least one therapeutic agent to a site in thelumen of a mammalian vessel, along with instruction means directing itsuse in accord with the present invention. In a preferred aspect, theinfusion catheter may be conveniently a double balloon or quadrupleballoon catheter with a permeable membrane. Preferably, the therapeuticagent comprises a cytoskeletal inhibitor.

It is also envisioned that the kit of the invention comprises anon-catheter delivery device, e.g., an adventitial wrap, a valve, stet,stent or shunt, for systemic or local delivery. A valve, stent, wrap orshunt useful in the methods of the invention can comprise abiodegradable coating or porous non-biodegradable coating, e.g., a PTFEmembrane, having dispersed therein one or more therapeutic agents of theinvention, preferably a sustained release dosage form of the therapeuticagent.

Another embodiment of the invention is a kit comprising a device adaptedfor the local delivery of at least two therapeutic agents, a unit dosageof a first therapeutic agent, and a unit dosage of a second therapeuticagent, along with instruction means directing their use in accord withthe present invention. The unit dosage forms of the first and secondagents may be introduced via discrete lumens of a catheter, or mixedtogether prior to introduction into a single lumen of a catheter. If theunit dosage forms are introduced into discrete lumens of a catheter, thedelivery of the agents to the vessel can occur simultaneously orsequentially. Moreover, a single lumen catheter may be employed todeliver a unit dosage form of one agent, followed by the reloading ofthe lumen with another agent and delivery of the other agent to thelumen of the vessel. Either or both unit dosages can act to reduce thediminution in vessel lumen diameter at the target site.

Alternatively, a unit dosage of one of the therapeutic agents may beadministered locally, e.g., via catheter, while a unit dosage of anothertherapeutic agent is administered systemically, e.g., via oraladministration.

The invention will be better understood by making reference to thefollowing specific examples.

EXAMPLE 1 Binding to Vascular Smooth Muscle Cells in the Blood VesselWall in Vivo

FIG. 1B illustrates the binding of NR-AN-01 (a murine IgG2b MAb) to thesmooth muscle cells in the vascular wall of an artery in a 24-year oldmale patient, 4 days after the i.v. administration of NR-AN-01. FIG. 1Bis a photomicrograph of a histological section taken through the medialregion of an arterial wall of the patient after NR-AN-01 administration,where the section was reacted ex vivo with HRP-conjugated goatanti-mouse IgG. The reaction of the HRP-conjugate with NR-AN-01 MAb wasvisualized by adding 4-chloro-1-naphthol or 3,3′-diaminobenzidinetetrahydrochloride as a peroxidase substrate (chromogen). The reactionproduct of the substrate forms an insoluble purple or dark brownprecipitate at the reaction site (shown at #2, FIG. 1B). A counter stainwas used to visualize collagenous extracellular matrix material (shownat #2, FIG. 1B) or cell nuclei (#1, FIG. 1B). Smooth muscle cells arevisualized under microscopic examination as purple stained cells (FIG.1A and FIG. 1B). This photomicrograph (FIG. 1B) demonstrates the abilityof the MAb to specifically bind to human vascular smooth muscle in vivo,and to be internalized by the cells and remain in the cells for extendedperiods.

EXAMPLE 2 Therapeutic Conjugates Containing Trichothecene TherapeuticAgents

Conjugates of NR-AN-01 and Roridin A were constructed by chemicallycoupling a hemisuccinate derivative of the trichothecene cytotoxin (asdescribed below) to a monoclonal antibody designated NR-AN-01. Twoconjugates were prepared, one coupled at the Roridin A 2′ position andone at the 13′ position. Two schemes were used in this synthesis, asdepicted in FIG. 2 and FIG. 3. The conjugate was then purified fromunreacted Roridin A by PD-10 SEPHAROSE® column chromatography(Pharmacia; Piscataway, N.J.), analyzed by size exclusion high pressureliquid chromatography, and the column fractions were characterized bySDS-PAGE and isoelectric focusing (IEF), as described below.

FIG. 2 shows diagrammatically the first reaction scheme for synthesis ofRoridin A hemisuccinyl succinimidate (RA-HS-NHS) through a two stepprocess with reagents: succinic anhydride, triethylamine (NEt₃) anddimethyl amino pyridine (DMAP) present in dichloromethane (CH₂Cl₂) atroom temperature (RT); and, N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) reagents also in CH₂Cl₂ at RT.

FIG. 3 shows diagrammatically the second reaction scheme for synthesisof Roridin A hemisuccinyl succinimidate (RA-HS-NHS) through a five stepprocess with reagents: t-butyl dimethyl silyl chloride (TBMS-Cl) andimidazole in dimethylformamide (DMF) at room temperature (RT); aceticanhydride, triethylamine (TEA), and diethylaminopyridine indichloromethane (CH₂Cl₂) at RT; succinic anhydride, triethylamine (TEA)and dimethylaminopyridine (DMAP) in (CH₂Cl₂) at RT; and,N-hydroxysuccinimide (NHS) and dicyclohexyl carbodiimide (DCC) reagents.

Synthesis of 2′ Roridin-A Hemisuccinic Acid (2):

To 0.5 g (0.94 mmol) of Roridin A, 15 ml of dichloromethane was added.To this solution with stirring was added 0.104 g (1.04 mmol) of succinicanhydride. To the reaction mixture, 0.2 ml of triethylamine in 5 mldichloromethane was added. To the homogeneous reaction mixture, acatalytic amount of dimethylaminopyridine was added and stirred at roomtemperature for 15 hours. Completion of the reaction was followed bythin layer chromatography (CH₂Cl₂:CH₃OH=9.7:0.3 with few drops of aceticacid). At the end of the reaction, 0.3 ml of glacial acetic acid wasadded and the solvent removed under reduced pressure. The dried cruderesidue was partitioned between water and methylene chloride. Thecombined methylene chloride extracts (3×50 ml) were dried over anhydroussodium sulfate, solvent was removed under vacuum and dried to yield0.575 g (96%) of a crude mixture of three compounds. Preparative C18HPLC separation of the crude mixture in 50% acetonitrile-water with 2%acetic acid yielded 0.36 g (60%) of 2 as a white solid.

Synthesis of Succinimidyl 2′-Roridin A Hemisuccinate (3):

To 0.3 g (0.476 mmol) of 2′ Roridin A hemisuccinic acid in 30 mldichloromethane, 0.055 g (0.478 mmol) N-hydroxysuccinimide was added. Tothe clear reaction mixture, 0.108 g (0.524 mmol)dicyclohexylcarbodiimide was added. The reaction mixture was stirred atroom temperature for 6 hours. Completion of the reaction was followed byTLC (CH₂Cl₂:CH₃OH=9.7:0.3 with a few drops of acetic acid) as adeveloping solvent. A few drops of glacial acetic acid was added to thereaction mixture and the solvent was removed under reduced pressure. Tothe dried residue dichloromethane was added and the precipitated DCU wasfiltered. Solvent from the filtrate was removed under reduced pressureto yield a white solid. From the crude product, 0.208 g (60%) of 3 waspurified by preparative HPLC in 50% acetonitrile with 2% acetic acid asa mobile phase.

Synthesis of 13′-t-Butyldimethylsilyl Roridin A (4):

To 72.3 mg (0.136 mmol) of Roridin A in 0.5 ml dimethylformamidesolution, 0.055 g (0.367 mmol) t-butyldimethylsilyl chloride and 0.025 g(0.368 mmol) of imidazole were added. The reaction mixture was stirredat room temperature for 15 hours. Completion of the reaction wasfollowed by silica gel thin layer chromatography using 1% MeOH—CHCl₃ asa developing solvent. Solvent from the reaction mixture was removed invacuo and dried. The crude product was partitioned between water andmethylene chloride. Solvent from the combined methylene chlorideextracts was removed under reduced pressure and dried. The crude productwas purified by flash chromatography using EtOAc:Hexane (1:3) as aneluting solvent. Solvent from the eluants was removed under reducedpressure to yield 0.66 g (75%) of 4 as a solid.

Synthesis of 13′-t-Butyldimethylsilyl 2′ Acetyl Roridin A (5):

To 0.1 g (0.155 mmol) of 13′-t-butyldimethylsilyl Roridin A in 10 mldichloromethane, 0.3 ml acetic anhydride, 0.2 ml triethylamine and a fewcrystals of dimethylaminopyridine were added and stored at roomtemperature for 2 hours. Completion of the reaction was followed by TLCin 1% methanol-methylene chloride as a developing solvent. Solvent wasremoved under reduced pressure and purified by a silica gel column using1% methanol-chloroform as an elution solvent. Solvent from the eluantswas removed under vacuum to yield 0.085 g (80%) of 5 as a solid.

Synthesis of 2′ Acetyl Roridin A (6):

To 0.05 g (0.073 mmol) of 2′ acetyl 13′-t-butyldimethylsilyl Roridin Ain 5 ml tetrahydrofuran, 0.3 ml of 1 M tetrabutyl-ammonium fluoridesolution in THF was added. The reaction mixture was stirred at roomtemperature for 2 hours. Completion of the reaction was followed bysilica gel thin layer chromatography using 1% MeOH—CHCl₃ as thedeveloping solvent. Solvent from the reaction mixture was removed underreduced pressure and dried. The crude product was purified on a silicagel column using 1% CH₃OH—CHCl₃ as an eluting solvent. Solvent from thecombined eluants were removed under vacuum to yield 0.020 g (48%) of 6as a solid.

Synthesis of 2′-Acetyl 13′-hemisuccinyl Roridin A (7):

To 0.05 g (0.087 mmol) of 2′-acetyl Roridin A in 1 ml ofdichloromethane, 0.025 g (0.25 mmol) succinic anhydride and 35 ml oftriethylamine was added. A few crystals of dimethylaminopyridine wasadded as a catalyst. The reaction mixture was stirred at roomtemperature for 24 hours. Completion of the reaction was followed bythin layer chromatography using 5% MeOH—CH₂Cl₂ as developing solvent. Atthe end of the reaction 30 ml of glacial acetic acid was added. Solventfrom the reaction mixture was removed under reduced pressure and dried.The crude product was partitioned between water and ethyl acetate.Solvent from the combined ethyl acetate fractions was removed underreduced pressure. Crude product was purified by passing through a silicagel column to yield 0.039 g (66%) of 7 as a white solid.

Synthesis of Succinimidyl 2′-Acetyl 13′-Roridin A Hemisuccinate (8):

To 0.036 g (0.0050 mmol) of 2′-acetyl 13′-Roridin A hemisuccinic acid in2 ml dichloromethane, 0.009 g (0.09 mmol) N-hydroxysuccinimide wasadded. To a stirred solution, 0.012 g (0.059 mmol)dicyclohexylcarbodiimide was added. The reaction mixture was stirred atroom temperature for 8 hours. Completion of the reaction was followed bysilica gel thin layer chromatography using 5% MeOH—CH₂Cl₂ as adeveloping solvent. A few drops of glacial acetic acid was added to thereaction mixture. Solvent from the reaction mixture was removed underreduced pressure and dried. The crude product was purified on a silicagel column using 5% MeOH—CH₂Cl₂ as an eluting solvent. Solvent from thecombined eluants was removed under vacuum to yield 0.025 g (61%) of 8 asa white solid.

Conjugation of Succinimidyl 2′-Roridin A Hemisuccinate (3) andSuccinimidyl 2′-Acetyl 13′-Roridin A Hemisuccinate (8) to NR-AN-01 WholeAntibody (MAb):

Conjugation reactions were performed at pH 8.0 in borate buffer in thepresence of 25% dimethylsulfoxide (DMSO) solvent at room temperaturewith gentle mixing for 45 minutes prior to purification by gelpermeation chromatography. The molar trichothecene drug precursor toantibody offerings were 25:1 and 40:1 for the 2′ and 13′ Roridin Aanalogues (3 and 8), respectively. Antibody concentration was 0.9 to 1.0mg/ml during the conjugation reaction.

A Typical 2′ Analogue (3) Reaction With 25 mg of Antibody was asfollows:

To 4.7 ml of 5.3 mg Ab/ml in phosphate buffered saline (i.e., PBS; 150mM NaCl, 6.7 mM Phosphate, pH 7.3) was added 10 ml PBS and 5 ml ofborate buffer (0.5 M, pH 8.0). With stirring gently to the reactionmixture, 6.3 ml of DMSO containing 1.37 mg of succinimidyl 2′ Roridin Ahemisuccinate (3) was then added dropwise over a 15 second period.

Purification:

To purify, one ml reaction aliquots were applied to Pharmacia PD-10Sepharose® columns equilibrated in PBS. The eluted conjugate wascollected in 2.4 to 4.8 ml fractions. The PD-10 purified conjugatealiquots were then pooled and concentrated on an Amicon PM-10 DiAflo®concentrator to 1.5 to 2.0 mg of Ab/ml; sterile filtered through a 0.2μGelman Acrodisc® and filled into sterile glass vials in 5 ml volume.

The 2′ conjugate was quick frozen in liquid nitrogen and then stored at−70° C. until use. The 13′ Roridin A NR-AN-01 conjugate was storedfrozen or refrigerated (i.e., 5-10° C.).

Characterization of Conjugates:

Protein concentration was determined by BCA assay using the copperreagent method (Pierce Chemical Corp.).

Assessment of degree of antibody derivatization was performed by firsthydrolyzing an aliquot of conjugate in 0.2 M carbonate, pH 10.3 for 4hours (at room temperature for 2′ conjugate or at 37° C. for the 13′conjugate) followed by filtration through a PM-30 membrane. The filtratewas then assayed for Roridin A on C-18 reverse phase HPLC using a mobilephase of 50:48:2 ratio CH₃CN:H₂O:HOAC, respectively. A 1.32 correctionfactor was used to correct for parallel macrocyclic ring decompositionthat gives polar products during the hydrolysis of the 13′ conjugate.

Size exclusion chromatography on DuPont Zorbax® HPLC and isoelectricfocusing using Serva® gel plates (pH 3 to 10) were also performed. Noindication of aggregation was observed by HPLC.

Immunoassay of the Roridin A-antibody conjugates was performed by eithercompetitive ELISA using biotinylated-Ab with Streptavidin/Peroxidasedetection or by a competitive cell binding assay using ¹²⁵I-labeledantibody. Alternatively, immunoreactivity was measured under conditionsof antigen saturation in a cell binding assay wherein antibody was firsttrace labeled with I-125 by the chloramine T method and thensubsequently derivatized with 2′ and 13′ Roridin A precursors.

EXAMPLE 3 Kinetics of Binding to Smooth Muscle Cells

For administration by i.v. catheter, it is desirable that thetherapeutic conjugates of the invention be administered in less than 3to 5 minutes, so that blood flow can be reestablished in the patient.Therefore, studies were conducted to determine the binding kinetics of asmooth muscle binding protein with a Ka of >10⁹ liter/mole. Becausehuman vascular smooth muscle cells grow slowly in culture, and baboonsmooth muscle cells were found to express the human CSPG cell surfacemarker, BO54 baboon artery smooth muscle cells and human A375 M/M(melanoma; ATCC #CRL1619) cells bearing CSPG surface marker were used inmany of the studies described in the Examples, below.

For the kinetic binding studies, A375 M/M and BO54 cells were seeded insterile 96 well microtiter plates at 2500 cells/well. Plates werewrapped in aluminum foil, and incubated at 37° C. overnight in ahumidified atmosphere of 5% CO₂/95% air. After approximately 18 hr,incubation plates were removed and cells were fixed with 0.05%glutaraldehyde for 5 minutes to prevent membrane turnover. Followingfixation, the plates were exhaustively washed with PBS containing 0.5%Tween-20®. Serial two-fold dilutions of an NR-AN-01 therapeuticconjugate containing Roridin A were prepared at protein concentrationsof 10 mg/ml to 20 ng/ml, and each dilution was aliquoted into two wells.The plates were incubated at 4° C. with the NR-AN-01 for 5, 15, 30, and60 minutes, after which the unbound protein was removed by aspirationand 100 ml of CS buffer was added (5% chicken serum/0.5% Tween-20® inPBS) to each well. CS buffer was removed and the NR-AN-01 therapeuticconjugate bound to the cells was visualized by adding 100 ml ofHRP-conjugated goat anti-mouse IgG (Sigma Chemical Co., St. Louis, Mo.)to each well; incubating at 4° C. for 1 hr.; washing with PBS/0.05%Tween® to remove unbound goat IgG; and, adding2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) chromogenicsubstrate (i.e., for HRP). After incubating for 30 minutes, the amountof NR-AN-01 bound to the cells was quantified by measuring theabsorbance at 415 nm and 490 nm using an ELISA plate reader equipped fordata acquisition by a Compaq computer.

FIG. 4A graphically depicts the results of in vitro studies in whichA375 m/m marker-positive cells were held at 4° C. (i.e., to preventmembrane turnover) for 5 minutes (open squares, FIG. 4A), 15 minutes(closed diamonds, FIG. 4A), 30 minutes (closed squares, FIG. 4A) or 60minutes (open diamonds, FIG. 4A) with different concentrations ofNR-AN-01 (NRAN01 μug/ml). The binding of the NR-AN-01 MAb to the A375cells was quantified by washing to remove unbound antibody, addingHRP-conjugated goat anti-mouse IgG to react with the cell-bound MAb,washing to remove unbound goat second antibody, and adding2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substratefor peroxidase. Color development was monitored after 30 minutes at both415 nm and 490 nm (ABS415,490).

FIG. 4B graphically depicts the results of in vitro studies conducted ina manner similar to those described above in regard to FIG. 4A, butusing BO54 marker-positive smooth muscle cells, i.e., instead of theA375 m/m cells.

The results presented in FIG. 4A and FIG. 4B show significant binding ofNR-AN-01 to A375 and BO54 cells within 5 minutes at 4° C., even at thelowest dose of 20 ng/ml.

EXAMPLE 4

Effects of Roridin A and RA-NR-AN-01 Conjugates The effects of Roridin A(RA) and RA-NR-AN-01 conjugates on cellular protein synthesis (i.e., by³H-leucine incorporation) and metabolic activity (i.e., by mitochondrialMTT assay) were tested in the experiments detailed in EXAMPLE 5 andEXAMPLE 6, below. The studies in EXAMPLE 4 include experiments todetermine the effects of long-term (i.e., 24 hour) treatment with theagents. The studies in EXAMPLE 5 include experiments to determine theeffects of “pulse” (i.e., 5 minute) treatment on cells. In both studies,the cellular specificity of the effects were evaluated by including“target” cells (i.e., cells bearing the CSPG “marker”) and non-targetcells. For comparative purposes, free-RA (i.e., uncoupled) was alsoincluded in the studies. The effects on cellular protein synthesis ormetabolic activity were evaluated either immediately following thetreatment, or a “recovery period” was allowed (i.e., involvingincubation of the cells overnight at 37° C.) to determine the long-termeffects of the agents on the cell populations.

Metabolic Effects After 24 Hours Exposure:

While it is known that monoclonal antibody-drug conjugates may have adegree of specificity for cells bearing marker antigens when employed invivo, it has proven more difficult in many systems to demonstrate invitro specificity of action, especially with compounds that arelipophilic. Therefore, the present experiments were conducted in whichthe inhibitory effects of the NR-AN-01-Roridin A conjugate was tested ontarget and non-target cells over 24 hours. The results with RA-NR-AN-01were compared to the effect of free Roridin A over the same 24-hourperiod. A modified methyl-tetrazolium blue (MTT) assay was utilized with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) todetermine cellular metabolic activity. This assay is thought to measurecellular mitochondrial dehydrogenase activity. For some of thesestudies, M14 (melanoma) and BO54 (smooth muscle) cell lines were used asmarker-positive target cells and HT29 cells (colon carcinoma; ATCC#HTB38) were used as the non-target specificity control. In otherstudies, A375 was used as a marker-positive cell. The HT29 and M14 cellswere seeded in 96-well microtiter plates at a concentration of 5.0×10³cells/well, and the BO54 cells were seeded at 2.5×10³ cells/well. Serialtwo-fold dilutions of free Roridin A and 2′RA-HS-NR-AN-01 (i.e., RoridinA coupled through a hemisuccinate (HS) coupling agent at the 2′ positionto NR-AN-01) were prepared in DMEM over a range of proteinconcentrations from 20 mg/ml to 40 pg/ml. Test agents were added (induplicate) to microtiter wells (100 ml/well), and the plates werewrapped in aluminum foil and incubated at 37° C. in a humidifiedatmosphere consisting of 5% CO₂/95% air for 24 hours. After 24 hours,medium was removed (by aspiration), fresh DMEM was added (100 ml/well),and the cells were returned to incubate for an additional overnight(i.e., 16-18 hours) “recovery period”. At the end of the “recoveryperiod” cellular metabolic activity was determined by adding 20 ml toeach well of a 5 mg/ml MTT solution. The plates were covered andincubated at 37° C. for 4 hours and then the reaction was developed byadding 100 ml/well of 10% SDS/0.1 N HCl. The dark blue solubilizedformazan reaction product was developed at room temperature after 16-18hours and quantified using an ELISA microtiter plate reader at anabsorbance of 570 nm.

FIG. 5A graphically depicts the results of in vitro studies in whichBO54 marker-positive smooth muscle cells were incubated with differentconcentrations of RA-NR-AN-01 (NRAN01-RA; open squares, FIG. 5A) or freeRoridin A (Free RA; closed diamonds, FIG. 5A) for a period of 24 hours,washed, and then returned to culture for an additional 16-18 hourovernight (o/n) recovery period prior to testing metabolic activity inan MTT assay. The concentrations of Free RA and RA-NR-AN-01 areexpressed as the calculated concentration of Roridin A (in mg/ml plottedon a log scale) in the assay (i.e., rather than the total mg/ml ofNR-AN-01 protein in the assay), so that direct comparisons could bemade. The metabolic activity of the cells in the MTT assay is presentedas the percentage of the metabolic activity measured in a controluntreated culture of cells (i.e., % control).

FIG. 5B graphically depicts the results of in vitro studies conducted ina manner similar to those described above in regard to FIG. 5A, butcomparing the effects of only RA-NR-AN-01 (NRAN01-RA) on three differentcell types: namely, BO54 marker-positive smooth muscle cells(BO54-NRAN01-RA; open squares, FIG. 5B); HT29 marker-negative controlcells (HT29-NRAN01-RA; closed diamonds, FIG. 5B); and, M14marker-positive cells (M14-NRAN01-RA; closed squares, FIG. 5B). Asdescribed above in regard to FIG. 5A, the concentrations in the presentexperiment are expressed in terms of ug/ml of Roridin A. Metabolicactivity of the cells is expressed in a manner similar to that in FIG.5A, i.e., as the percentage of activity measured in an untreated controlculture of cells (% control).

The results presented in FIG. 5A and FIG. 5B show that metabolicactivity measured in the MTT assay was significantly decreased in allpopulations of test cells, even 16-18 hours after a 24-hour incubationin either free Roridin A or the 2′ or 13′ RA-NR-AN-01 conjugates. Theeffects of the RA-NR-AN-01-conjugates appeared to be non-specificallyinhibitory for both target (BO54 and M14) and non-target (HT29) cells(FIGS. 5A and 5B). The inhibitory effects were observed at a freeRoridin A or RA-conjugate concentration of >10 ng/ml.

For comparative purposes, a second study was conducted in which theeffects of Pseudomonas exotoxin (PE) conjugates on cells were evaluatedin a similar protocol. For these studies, target and non-target cellswere treated with PE or PE-NR-AN-01 for 24 hours, and then allowed a“recovery period” (as above) before metabolic activity was tested in anMTT assay.

FIG. 6A graphically depicts the results of in vitro studies conducted ina manner similar to those described above in regard to FIG. 5A, butdesigned to study the metabolic effects of PE-NR-AN-01 (NRAN01-PE) oncells, i.e., rather than RA-NR-AN-01. Three different cell types wereutilized: namely, BO54 marker-positive smooth muscle cells (BO54; opensquares, FIG. 6A); HT29 marker-negative control cells (HT29; closeddiamonds, FIG. 6A); and, M14 marker-positive cells (MT14; closedsquares, FIG. 6A). In this study, the concentration of conjugate isexpressed in μg/ml NR-AN-01 protein (plotted on a log scale), and themetabolic activity is expressed as the percentage of the MTT activitymeasured in an untreated control culture (% control).

FIG. 6B graphically depicts the results of in vitro studies conducted inmanner similar to those discussed above in regard to FIG. 6A, butdesigned to compare the effects obtained with free PE (PE) to thoseobtained above, i.e., in FIG. 6A, with PE-NR-AN-01. The cells, cultureconditions, calculations, and presentation of the results are the sameas in FIG. 6A, above.

The results presented in FIG. 6A and FIG. 6B show that 24 hours exposureto PE-NR-AN-01 or free PE was non-specifically inhibitory to cells atconcentrations of >100 ng/ml.

While this type of non-specific inhibition was judged to be of potentialvalue for biological atheroectomy, it was not considered desirable fortreatment of restenosis following angioplasty where dead and dying cellsmay release factors that stimulate smooth muscle proliferation.

EXAMPLE 5 Effects of Pulse-treatment on Cellular Activity

Additional studies were conducted to evaluate the effects of ashort-term, i.e., 5 minute, exposure to a Roridin A-containingtherapeutic conjugate on cells. In these studies, both metabolicactivity (measured in MTT assays) and cellular protein synthesis(measured by ³H-leucine incorporation) were evaluated.

Effects After 5 Minutes of Exposure: Protein Synthesis

The effects of a 5-minute exposure to free Roridin A (RA) or atherapeutic conjugate were evaluated. Roridin A-NR-AN-01 coupled througha hemisuccinyl (HS) at either the 2′ position (2′RA-HS-NR-AN-01) or the13′ position (13′RA-HS-NR-AN-01) were employed. (In the case of13′RA-HS-NR-AN-01, the 2′ position of Roridin A was also acetylated.)The RA, 2′ or 13′RA-NR-AN-01 conjugates were diluted two fold in sterileDMEM over a range of concentrations from 400 ng/ml to 780 pg/ml ofRoridin A. (The test samples were all normalized to Roridin A, so thatdirect comparisons could be made of the effects at comparable doses.)Samples were aliquoted (in duplicate) into duplicate microtiter platesat 100 ml/well and incubated at room temperature for five minutes.

Both short-term and long-term effects of the test samples onmarker-positive A375 and marker-negative HT29 cells were determined. Forstudying the short-term effects, 100 ml/well of [³H]-leucine (0.5mCi/ml) was added immediately after the 5-minute treatment withconjugate (or RA) and protein synthesis was evaluated over a four-hourperiod. For determining the long-term effects, the cells were treatedfor 5 minutes, washed, and then returned to culture for a 24-hour“recovery” period in DMEM medium containing either 5% NBS/5% Serum Plus®(i.e., for A375 or HT29 cells) or 10% FBS (i.e., for BO54 cells). At theend of the “recovery” period, the incubation medium was removed (i.e.,by aspiration) and ³H-leucine was added (as above). In both cases (i.e.,whether short-term or long-term), protein synthesis of the cells wasevaluated by incubating the cells with the ³H-leucine for 4 hours at 37°C. in a humidified chamber (as above), and all results are calculated bycomparison with non-treated cells (i.e., 100% control). After 4 hoursthe ³H-leucine was removed, the cells were removed from the substrata bytrypsin-treatment, aspirated (using a PHD™ cell harvester (CambridgeTechnology, Inc., Cambridge, Mass.)) and collected by filtration onglass fiber filters. The glass fiber filters were dried andradioactivity quantified by liquid scintillation spectroscopy in aBeckman liquid scintillation counter.

FIG. 7A graphically depicts the results of in vitro studies conducted toinvestigate the effects on control HT29 marker-negative cells of a 5minute exposure to different concentrations of Roridin A (Free RA; opensquares, FIG. 7A), or 2′RA-NR-AN-01 (2′RA-NRAN01; closed squares, FIG.7A), or 13′RA-NR-AN-01 (13′RA-NRAN0 1; closed triangles, FIG. 7A)conjugates. The concentrations of Free RA, 2′RA-NR-AN-01 or 13′NR-AN-01are expressed as the calculated concentration of Roridin A in the assay(in μg/ml plotted on a log scale), i.e., rather than the total μg/ml ofNR-AN-01 protein, so that direct comparisons of the results can be made.For these studies, the cells were treated for 5 minutes, washed, andthen returned to culture for 4 hours, during which time cellular proteinsynthesis was evaluated by adding 0.5 mCi/ml of ³H-leucine to theculture medium. At the end of the 4 hour period, cellular proteins werecollected and radioactivity was determined. The results are expressed asthe percentage of the radioactivity recorded in a control (non-treated)HT29 cell culture (i.e., %control).

FIG. 7B graphically depicts the results of in vitro studiesinvestigating the effects on control HT29 marker-negative cells of a 5minute expose to different concentrations of Free RA (open squares, FIG.7B), 2′RA-NRAN01 (closed squares, FIG. 7B), or 13′RA-NRAN01 (closedtriangles, FIG. 7B), as described above in regard to FIG. 7A, but in thepresent experiments the cells were incubated for a 16-18 hour recoveryperiod (i.e., overnight; o/n) prior to testing protein synthesis in afour hour ³H-leucine protein synthesis assay. The results are presentedin a manner similar to those above in FIG. 7A.

The results presented in FIG. 7A and FIG. 7B show the short-term andlong-term effects, respectively, of RA, 2′RA-HS-NR-AN-01, and13′RA-HS-NR-AN-01 on protein synthesis by HT29 control cells. Theresults show a dose-response inhibition of cellular protein synthesis bythe free Roridin A, but not by RA-NR-AN-01, in HT29 cells. Theinhibition triggered by RA during the 5 minutes of incubation was stillmanifest after the 16-18 hours recovery period (FIG. 7B). In contrast,treatment of non-target HT29 cells with 2′RA-HS-NR-AN-01 or13′RA-HS-NR-AN-01 did not result in detectable inhibition of proteinsynthesis. Thus, these results (in contrast to those obtained above over24 hours) seem to suggest a surprising degree of specificity to the invitro action of the NR-AN-01-conjugates when treatment was delivered ina 5-minute “pulse”. However, it was also possible that theNR-AN-01-conjugate was inactive, and so additional experiments wereconducted to evaluate the effect of the conjugates on target cells.

FIG. 7C graphically depicts the results of in vitro studiesinvestigating the effects on A375 m/m marker-positive cells of a 5minute exposure to different concentrations of Free RA (open squares,FIG. 7C), 2′RA-NR-AN-01 (closed squares, FIG. 7C) or 13′RA-NR-AN-01(closed triangles, FIG. 7C), as described above in regard to FIG. 7A. Inthe present studies, the A375 cells were incubated for 5 minutes in thetest agent, washed, and tested for protein synthesis over the next 4hours by adding 0.5 mCi/ml ³H-leucine to the culture medium. The resultsof the experiments are plotted in a manner similar to those described,above, in regard to FIG. 7A.

FIG. 7D graphically depicts the results of in vitro studiesinvestigating the effects on A375 m/ml marker-positive cells of a 5minute exposure to different concentrations of Free RA (open squares,FIG. 7D), 2′RA-NRAN01 (closed squares, FIG. 7D), 13′RA-NRAN01 (closedtriangles, FIG. 7D), as described above in regard to FIG. 7B. In thepresent studies, the A375 cells were incubated for 5 minutes in the testagent, washed, and then returned to culture for a 16-18 hour recoveryperiod (i.e., overnight; o/n Recovery), after which time proteinsynthesis was evaluated during a 4 hour ³H-leucine protein synthesisassay. The results of the experiments are plotted in a manner similar tothose described above in regard to FIG. 7A.

The results presented in FIG. 7C and FIG. 7D show the short-term andlong-term effects, respectively, of RA, 2′RA-HS-NR-AN-01 and13′-RA-HS-NR-AN-01 on protein synthesis by A375 target cells. Treatmentof target cells with either the 2′ or 13′RA-NR-AN-01 therapeuticconjugate resulted in a short-term inhibition of protein synthesis,i.e., observed immediately after the 5-minute pulse treatment (FIG. 7C).These findings, when combined with the findings in FIG. 7A and FIG. 7B,above, suggest that the RA-NR-AN-01 conjugates were active and that theywere specifically inhibitory for target cells but not non-target cells.Interestingly, when “pulse” treated target cells were returned toculture no long-term inhibitory effects were observed (FIG. 7D). Theresults presented in FIG. 7C and FIG. 7D again show that Roridin A isnon-specifically inhibitory to test cells (i.e., in a manner similar toFIG. 7A and FIG. 7B, above) and that its effect on the cells is manifesteven after a 16-18 hour recovery period. Thus, the specific effects ofthe RA-NR-AN-01 conjugates on target cells during a “pulse” treatmentappear to be a property of the NR-AN-01 binding protein.

The results obtained with BO54 arterial smooth muscle cells were similarto those obtained with the A375 cells, above, i.e., free Roridin Ashowed a dose-response inhibition of protein synthesis in the short-termequated to be 60%, 66%, and 90% of control at 200 ng/ml, 100 ng/ml, and50 ng/ml; and in long-term the effects on protein synthesis were equatedto be 27%, 46%, and 98% of control at the same dosages. In contrast, the2′ or 13′RA-NR-AN-01 showed only 10-20% inhibition for short- orlong-term effects on protein synthesis (i.e., >80% of control).

Thus, the results show a short-term specific reversible effect ofRoridin A-conjugated NR-AN-01 on target cells when delivered as a“pulse” treatment. However, since only protein synthesis was evaluatedin these experiments, it was possible that cellular metabolic activitymight be affected in the cells as a result of the “pulse” treatment.Therefore, additional studies were conducted in which cellular metabolicactivity was evaluated following “pulse” treatment.

Effects After 5 Minutes of Exposure: Metabolic Activity

MTT assays were conducted at 48 hours following a 5-minute exposure oftarget and non-target cells to RA or RA-NR-AN-01 conjugates. Targetcells in these studies included BO54 and A375, and non-target cellsincluded HT29 cells. Sterile 96 well microtiter plates were seeded with2500 cells/well, wrapped in aluminum foil and incubated in a humidifiedchamber containing 5% CO₂/95% air for 16-18 hours. Serial two-folddilutions of Roridin A (RA), 2′RA-HS-NR-AN-01 and 13′RA-HS-NR-AN-01 wereprepared from 400 ng/ml to 780 pg/ml, and 100 ml aliquots of thedilutions were dispensed into duplicate wells. After 5 minutes exposureto the test samples, the cells were washed to remove the test samples,and fresh medium was added. The cells were allowed 48 hours of recoveryprior to testing: i.e., plates were incubated for 48 hours, and thencellular metabolic activity was determined by adding 20 ml/well of a 5mg/ml MTT solution. The plates were covered and incubated at 37° C. for4 hours and then the reaction was developed as described above (seeEXAMPLE 4, above). The dark blue solubilized formazan reaction productwas developed at room temperature after a 16-18 hour incubation. Thesamples were quantified using an ELISA microtiter plate reader at anabsorbance of 570 nm.

FIG. 8A graphically depicts the results of in vitro studiesinvestigating the effects on BO54 marker-positive smooth muscle cells ofa 5 minute exposure to different concentrations of Roridin A (opensquares, FIG. 8A), 2′RA-NR-AN-01 (NRAN01-2′RA; closed diamonds, FIG.8A), or 13′ RA-NR-AN-01 (NRAN01-13′RA; closed squares, FIG. 8A). Theexperiments were conducted in a manner similar to those described abovein regard to FIG. 7B, but metabolic activity was assayed by MTT assay,i.e., rather than protein synthesis as in FIG. 7B, and cells were alsogiven 48 hours to recover (rather than 24 hours, as in FIG. 7B). Theresults of the experiments are plotted in a manner similar to hosedescribed (above) in regard to FIG. 7A.

FIG. 8B graphically depicts the results of in vitro studiesinvestigating the effects on A375 m/m marker-positive cells of a 5minute exposure to different concentrations of Roridin A (open squares,FIG. 8B), 2′RA-NR-AN-01 (NRAN01-2′RA; closed diamonds, FIG. 8B),13′RA-NR-AN-01 (NRAN01-13′RA; closed squares, FIG. 8B). The experimentswere conducted (and the results plotted) in a manner similar to thosedescribed above in regard to FIG. 8A.

FIG. 8C graphically depicts the results of in vitro studiesinvestigating the effects on HT29 marker-negative cells of a 5 minuteexposure to different concentrations of Roridin A (open squares, FIG.8C), 2′RA-NR-AN-01 (NRAN01-2′RA; closed diamonds, FIG. 8C),13′RA-NR-AN-01 (NRAN01-13′RA; closed squares, FIG. 8C). The experimentswere conducted (and the results plotted) in a manner similar to thosedescribed above in regard to FIG. 8A.

The results presented in FIGS. 8A-8C show slight differences between thedifferent RA-NR-AN-01 conjugates at the highest doses, but at the lowerdoses the 2′ and 13′RA-NR-AN-01 did not significantly inhibit targetcell (i.e., BO54 and A375) or non-target cell (i.e., HT29) metabolicactivity over the long-term (i.e., 48 hours). Thus, the results suggestthat the short-term inhibition of target cell protein synthesis (FIGS.7C-7D, above) does not result in long-term metabolic effects on thecells, as measurable in MTT assays. That these assays were able todetect metabolic alterations in cells resulting from a 5 minute exposureis evidenced by the results obtained with free Roridin A. In this case,free Roridin A was non-specifically inhibitory to target and non-targetcell types, even when the cells were exposed to the agent for only 5minutes and then returned to culture for the 48-hour recovery period(FIGS. 8A-8C).

Thus, the findings with free Roridin A suggest that the MTT assay wascapable of detecting metabolic alterations induced during a 5-minuteexposure. Taken together these finding suggest that RA-NR-AN-01conjugates can specifically inhibit target cell activity (i.e., proteinsynthesis) when administered in a “pulse” treatment, and that theseeffects were reversible without significant long-term effects on eitherprotein synthesis or cellular metabolic activity (as measured in an MTTassay). These in vitro properties of the RA-NR-AN-01 conjugates werejudged to be highly useful for inhibition of smooth muscle cell activityin vivo. Therefore, animal model studies were next conducted to evaluatethe effects of these therapeutic conjugates in vivo.

EXAMPLE 6 Determination of Infusion Conditions in an Animal Model

The therapeutic conjugates of the invention are useful for inhibitingstenosis following vascular trauma or disease. In an illustrativeexample, vascular trauma that is induced during angioplasty is treatedduring the surgical procedure by removing the catheter used to performthe angioplasty, and inserting a balloon infusion catheter into thevessel. The infusion catheter is positioned with the instillation port(or, alternatively, a permeable membrane region) in the traumatized areaof the vessel, and then pressure is applied to introduce the therapeuticconjugate. For example, an infusion catheter with two balloons may beused, and when one balloon is inflated on either side of the trauma sitea fluid space is created that can be filled with a suitable infusionfluid containing the therapeutic conjugate. It has been reportedpreviously that infusion of a horseradish peroxidase (HRP) marker enzymeat a pressure of 300 mm Hg over 45 seconds in dog or human coronaryarteries resulted in penetration of the HRP into the vessel wall(Goldman et al., supra). However, HRP is a smaller molecule thanNR-AN-01 and human and dog coronary arteries are also considerablysmaller than the carotid or femoral arteries in the present domestic pigmodel system. Experiments were therefore conducted to determine, in adomestic pig model system, the infusion conditions suitable for deliveryof a therapeutic conjugate to the vascular smooth muscle cells incarotid and femoral arteries. Delivery conditions were monitored byevaluating the penetration of the therapeutic conjugate into thevascular wall, and specific binding of the therapeutic conjugate to thevascular smooth muscle cells in the vessel wall.

Using an infusion catheter, the coronary and femoral arteries ofdomestic pigs or non-human primates were infused with NR-AN-01 for 45seconds to 3 minutes at multiple pressures in the range of about 0.4atmospheres (300 mm Hg) to 3 atmospheres. After infusion, the vesselswere flushed with sterile saline and prepared for immunohistochemistryusing HRP-conjugated goat anti-mouse IgG to detect the NR-AN-01 mouseIgG in the vessel wall. It was determined that full penetration wasachieved of NR-AN-01 into these vessel walls at a pressure of 3atmospheres after 3 minutes.

Immunohistology was also used to determine which animal model systemsexpressed the target antigen for NR-AN-01. Vascular tissue sections fromreadily available experimental animal species were exposed to NR-AN-01,washed, and reacted with HRP-conjugated goat anti-mouse IgG. Onlynon-human primates and swine were found to share the 250 kD NR-AN-01target antigen with man.

To determine whether NR-AN-01 could bind in a specific manner to itstarget antigen in vivo, the coronary and femoral arteries of domesticpigs were infused with therapeutic conjugates using an infusioncatheter, the infusion sites were flushed with sterile saline, thesurgical sites were then closed, and the animals were maintained for anadditional 3-5 days. At the end of this time, the vascular infusionsites were excised and prepared for immunohistology, once again usinggoat anti-mouse IgG to identify NR-AN-01. NR-AN-01 was identified in thevessel wall of swine coronary and femoral arteries 3-5 days aftersurgery, and the NR-AN-01 appeared to be associated only with vascularsmooth muscle cells. These findings suggest that NR-AN-01 is capable ofspecifically binding to its target antigen in vivo.

EXAMPLE 7 Inhibition of Vascular Smooth Muscle Cells In Vivo

Intimal smooth muscle proliferation that follows ballooncatheter-induced trauma is a good model to evaluate the therapeuticefficacy of conjugates for inhibiting smooth muscle cell activity invivo in response to vascular trauma, including restenosis followingangioplasty. Domestic pigs were used to study the effects of NR-AN-01(i.e., termed vascular smooth muscle binding protein or simply VSMBP inthese studies; and therapeutic conjugates with Roridin A are termedVSMBP-RA). The events which normally follow balloon angioplasty in theporcine artery have been described previously (Steele et al., Circ.Res., 57: 105-112 (1985)). In these studies, dilation of the carotidartery using an oversized balloon (balloon:artery ratio approximately1.5:1) resulted in complete endothelial denudation over an area of 1.5-2cm in length. Although this length of traumatic injury was selected inan attempt to minimize thrombosis, there was still marked plateletdeposition and thrombus formation. The procedure also resulted indissection through the internal elastic lamina into the arterial mediaand necrosis of medial smooth muscle cells. Intimal thickening due tosmooth muscle proliferation was apparent 7 days after injury and reacheda mean maximum thickness of 85 mm at 14 days. The histologicalappearance of this neointima is very similar to the proliferativeneointimal tissue of human restenosis (Schwartz et al., Circ., 82:2190-2200 (1990)).

A single dose test protocol was conducted in domestic pigs withNR-AN-01-Roridin A conjugates. Localized administration of the testconjugates, i.e., through a catheter into a region of traumatized vesselconfined by temporary slip ligatures, was designed to reduce systemictoxicity while providing a high level of exposure for the target smoothmuscle cells. This intra-artery route of administration in animal modelstudies simulates the proposed route in human coronary arteries. Thetest protocol was designed as an initial in vivo screening ofintra-arteriolar, site specific, catheter administered, vascular smoothmuscle binding protein (VSMBP) conjugates.

Toxicity of free drug was also evaluated, i.e., for pathobiologicaleffects on arteriolar smooth muscle cells. The therapeutically effectivedosage of the Roridin A-NR-AN-01 conjugate was determined by in vitrostudies, and the proper intra-arteriolar administration pressure wasdetermined by administering free MAb and MAb conjugates to animals, asdescribed above in Example 7.

Six domestic crossbred swine (Duroc X), weanling feeder pigs ofapproximately 30 pounds body weight, were used in the experiment. Theanimals were randomly assigned to the following treatment regimen whereeach pig has four different treatments divided between the right andleft carotid and femoral arteries, one of which is a PBS control (Tables1-3, below).

TABLE 1 GROUP TREATMENT NO. GROUP MATERIAL DESCRIPTION 1 CONTROL, VSMBP,200 μg/ml in PBS, pH 6.5 VSMBP 2 CONTROL, PBS PBS, pH 6.5, in injectionsterile water 3 CONTROL, DRUG Roridin A, 2.5 μg/ml in PBS, pH 6.5 4TEST, VSMBP-RA2′ (200 μg/ml VSMBP & CONJUGATE 2.5 μg/ml RA) 5 TEST,VSMBP-RA13′ (200 μg/ml VSMBP & CONJUGATE 3.1 μg/ml RA) 6 TEST, CONJ + RAVSMBP-RA2′ (200 μg/ml VSMBP & 2.5 μg/ml RA) PLUS free Roridin A (2.5μg/ml) 7 TEST, CONJ + RA VSMBP-RA13′ (200 μg/ml VSMBP & 3.1 μg/ml RA)PLUS free Roridin A (2.5 μg/ml)

Surgical Procedure:

Test conjugates and control compounds were administered as a singleintra-artery infusion at the site of endothelial denuding and traumainduced by a balloon catheter. Both the carotid and femoral arterieswere abraded over 1 cm to 2 cm of endothelium by intraluminal passage ofa 23 cm, size 3 (femoral) and size 4 (carotid) Uresil Vascu-Flo®silicone occlusion balloon catheter (Uresil Technology Center, Skokie,Ill.), sufficiently distended with saline to generate slight resistance.This technique produced slight distension of the artery. Following thistreatment, proximal and distal slip ligatures, 3-0 silk, were placednear the ends of the abraded region, and a size 8 French, Infant FeedingCatheter (Cutter-Resi flex, Berkeley, Calif.) attached to an InflationPro® (USCI, C.R. Bard, Inc., Billerica, Mass.) pressure syringe was usedto administer the test conjugates and control compounds directly to thedenuded segment at a pressure of three atmospheres for three minutes.The slip ligatures were removed after the three minute exposure periodand arterial blood flow was re-established. In these studies, branchesof the femoral or carotid arteries were ligated with 00 silk suture asrequired to attain pressurized infusion in the treated region. Thelargest distal branch of the femoral artery (the saphenous artery) wasincised and used as an entry site for the catheters which were thenpassed into the main femoral artery. Following this catheterizationprocedure in the main femoral artery, the secondary branch was ligated.In these cases, ligation or incision was used to allow entry of thecatheters and the opening was then closed with 3 to 4 sutures of 5-0monosilamen polybutester (Novafil, D & G Monofil Inc., Monati, PR).

Follow-up Procedures:

Following surgery, the pigs were kept in 3×5 foot indoor runs withcement floors during the quarantine and surgical recovery periods. Theywere then transferred to indoor/outdoor pens for the remainder of thefive week healing period prior to collection of tissues for histology.

The animals recovered normally from surgery with no evidence ofhemorrhage or inflammation at the surgical sites. All six animals wereexamined 5 to 6 days after treatment with a doppler stethoscope, and allarteries in each of the animals were patent. Post treatment all animalshad normal appetite, activity and weight gain.

Gross Pathology and Histological Evaluation:

Five weeks following the traumatization and treatment of the arteries,the animals were sedated with 0.6 ml Telazol® (tiletamine hydrochloride;A.H. Robins Co., Richmond, Va.) and 0.5 ml xylazine (Lloyd Laboratories,Shenandoah, Iowa) per 30 lb body weight by intramuscular injection,heparinized (i.v. 2 ml sodium heparin, 1000 units/ml), and euthanized byi.v. pentobarbital. Both the right and left carotid and femoral arterieswere removed with normal vessel included both proximal and distal to thetreated segment. The arteries were measured and the location ofligatures and gross abnormalities noted. The arteries were transected at2 mm intervals and arranged in order in cryomolds with O.C.T. (optimumcutting temperature) compound (Tissue Tek®, Miles Laboratories Inc.,Elkhart, Ind.) and frozen in liquid nitrogen. The blocks were sectionedat 5 microns and stained with H&E, Massons Trichrome and MovatsPentachrome for morphological studies. Sections were also used forimmunohistological staining of vascular smooth muscle.

Histological examination of the step sections of the arteries revealedmarked inhibition of intimal smooth muscle proliferation in the regionstraumatized and treated with RA-NR-AN-01 conjugates (Table 2). Thisinhibition was evident even at sub-gross evaluation of the vessels. Theinhibition of intimal smooth muscle cell proliferation was produced withminimal or no histological evidence of smooth muscle cell death in theartery wall. A cross-sections of one such traumatized artery is providedin FIGS. 9A and 9B.

TABLE 2 INTIMAL SMOOTH MUSCLE PROLIFERATION IN TRAUMATIZED AND TREATEDPORCINE ARTERIES NO. INTIMAL SMC ARTERIES HYPERTROPHY* TREATMENTEVALUATED ave. (range) Control, MAB 4 3.75 (3-4) Control, PBS 4 4 (4)Control, RA 2 4 (4) Test, 2′RA (High pressure) 1 1 (1) (Low pressure) 13 (3) Test, 13′RA (High pressure) 1 1 (1) (Low pressure) 1 1 (1)*Intimal SMC Hypertrophy: intimal smooth muscle cell hypertrophy scoredon a scale from 1+ (minimal) to 4+ (maximal).

The results presented in FIG. 9A show (at 160× magnification) across-sectional of an untreated artery 5 weeks after angioplasty.Dominant histological features of the artery include displacement of theendothelium (see #1 in FIG. 9A) away from the internal elastic lamina(see #2, FIG. 9A), apparently due to intimal smooth muscle proliferation(see #3, FIG. 9A).

The results presented in FIG. 9B show (at 160× magnification) across-section of a treated artery 5 weeks after angioplasty and infusionof the RA-NR-AN-01 therapeutic conjugate. The vessel in this section wassubjected to greater mechanical stresses than the vessel shown in FIG.9A, with multiple sites where the external elastic membrane was rupturedand associated proliferation of smooth muscle cells in the outer layersof the media was observed (i.e., see #4 in FIG. 9B). Treatment withtherapeutic conjugate inhibited intimal hypertrophy, as evidenced by thelack of displacement of the endothelium (see #1, FIG. 9B) from theinternal elastic lamina (see #2, FIG. 9B). Surprisingly, this inhibitoryeffect on intimal smooth muscle cells was accomplished withoutinhibiting hypertrophy of medial smooth muscle cells in the areas wherethe external elastic membrane was ruptured (see #4, FIG. 9B).

This is a highly fortunate result because wound healing proceeds in thetreated vessel without the adverse consequences of intimal hyperplasiaand stenosis, or necrosis of smooth muscle cells in the media.

In these histological studies, comparisons were also made of theeffectiveness of both the 2′ and the 13′-Roridin A conjugate with thefinding that the 13′ conjugate (i.e., 13′RA-HS-NR-AN-01) appeared to bemore active in inhibiting intimal hyperplasia of smooth muscle cellsthan the 2′ conjugate (i.e., 2′RA-HS-NR-AN-01). In this study, lowpressure infusion of the 13′ conjugate appeared to inhibit smooth muscleproliferation more effectively than high pressure and the 13′ conjugatealso appeared to be more effective than the 2′ conjugate.

In FIG. 9B, therapeutic conjugate administered at the site followingangioplasty resulted in approximately 95% inhibition of the smoothmuscle hypertrophy that restricted the lumen of the untreated vessel(FIG. 9A). Significantly, the therapeutic conjugate exerted its effectson the smooth muscle cells migrating from the medial smooth musclelayers into the intima, without affecting either endothelium, orproducing any signs of necrosis (i.e., cell death) in the smooth musclecells in the medial layers of the arterial wall. Studies also failed toshow any histological signs of mononuclear infiltration or fibrosis suchas might result from toxic effects on the vessel wall. Also, visiblesigns of healing were observed in the intimal layers of treated vesselsand with re-growth of endothelium observed, i.e., endothelial cellsgrowing over the thin layer of smooth muscle cells in the intima thatlie between the endothelium and internal elastic lamina (i.e., #1 and#2, FIG. 9B). These combined histological observations suggest thehighly desirable features of wound healing, re-growth of endothelium andimproved vascular strength following treatment with a therapeuticconjugate that inhibits smooth muscle hyperplasia in the intimal layersof the vessel.

EXAMPLE 8 Vascular Smooth Muscle Cell in Vitro DNA and Protein SynthesisInhibition

The ability of various therapeutic agents to inhibit DNA synthesis andprotein synthesis in vascular smooth muscle cells was tested. ³H-leucineand ³H-thymidine uptake and cytotoxicity assays were conducted inaccordance with the following protocols.

minute exposure; ³H-leucine uptake: Vascular smooth muscle cells at40,000 cells/ml were seeded in sterile 24 well plates at 1 ml/well. Theplates were incubated overnight at 37° C., 5% CO₂, 95% air in ahumidified atmosphere (saturation). Log dilutions of the therapeuticagent of interest were incubated with the vascular smooth muscle cellsfor 5 minutes or 24 hours. Samples of the therapeutic agents werediluted in DMEM:F-12 medium (Whittaker Bioproducts, Walkersville,Maryland) with 5% fetal bovine serum (FBS, Gibco BRL, Gaithersburg, Md.)and 5% Serum Plus® (JRH Biosciences, Lenexa, Kans.). Followingtherapeutic agent incubation, the solution was aspirated, and 1 ml/wellof 0.5 microcurie/ml ³H-leucine in leucine-free DMEM (Dulbecco'sModified Eagle's Medium) with 5% Serum Plus® was added. The plates werere-incubated overnight at 37° C., 5% CO₂ in a humidified atmosphere. Thecells were visually graded using an inverted microscope using a scoringscale to determine viability and cell number. The 1 to 3 grade is basedupon percent of cell viability and number compared to control wells,with 3=100%, 2=70%-100% and 1=0%-70%. A record of this scoring assistedin determining the immediate cytotoxic effect of the therapeutic agents.The medium was then aspirated, and the cells were washed twice with cold5% TCA. 400 microliters of 0.2 M NaOH was added per well, and the plateswere incubated for two hours at room temperature on a rotating platform.200 microliters per well of the cell solution was transferred intoplastic scintillation vials (Bio-Rad Laboratories), and 4 milliliters ofBio-Safe® II liquid scintillation fluid (Research Products InterCorp.,Mount Prospect, Ill.) was added prior to vortexing. Vials were countedon a Beckman LS2800 liquid scintillation counter interfaced with Beckman“Data Capture” software for conversion to a Lotus 1-2-3® file andanalysis using Lotus 1-2-3®.

minute exposure; ³H-thymidine uptake: Vascular smooth muscle cells wereincubated in complete medium with 5% FBS (Gibco) overnight at 37° C. ina humidified, 5% CO₂ environment in sterile 24 well plates. The mediumwas aspirated from the wells and serum free medium supplemented withgrowth factors (DMEM: F-12 basal medium supplemented with growth factorcocktail, catalog number I1884, which contains insulin (5micrograms/ml), transferrin (5 micrograms/ml) and sodium selenite (5nanograms/ml), available from Sigma Chemical, St. Louis, Mo.) was added.Cells were incubated in this medium for 24 hours. For a 5 minutetherapeutic agent exposure, log dilutions of the therapeutic agent wereincubated with the cells in complete medium. After 5 minutes and mediumaspiration, 1 ml/well of 1.0 microcurie/ml ³H-thymidine dispersed incomplete medium was added. The 24 hour exposure involved incubation ofthe cells with 1 ml/well of 1.0 microcurie/ml of ³H-thymidine dispersedin complete medium and log dilutions of the therapeutic agent beingtested. In both exposure trials, the cells were then incubated overnightat 37° C. in a humidified, 5% CO₂ environment. The cells were visuallyscored for viability and cell number. Cells were washed and prepared fortransfer into plastic scintillation vials as described for the³H-leucine protocol. Vials were counted on a Beckman LS2800 liquidscintillation counter interfaced with Beckman “Data Capture” softwarefor conversion to a Lotus 1-2-3® file and analysis using Lotus 1-2-3®.

These protocols are amenable to use with other target cell populations,especially adherent monolayer cell types.

Morphological Cytotoxicity Evaluation-Pulsed Exposure: Vascular smoothmuscle cells were seeded at 4.0×10⁴ cells/ml medium/well on acommercially prepared four well slide (Nunc, Inc., Naperville, Ill.).Enough slides were seeded to accommodate two pulsed exposure lengths (5minutes and 24 hours) and prescribed increment evaluation points (24hours to 128 hours). All slides were run in duplicate to reveal anyassay anomalies. The therapeutic agent was diluted in the same mediumused in the ³H-leucine and ³H-thymidine assays. Each four well slide wasconcentration bracketed to one log greater concentration (well “B”), onelog lower concentration (well “D”) of the minimal effectiveconcentration (well “C”), as determined by the 3H-leucine and3H-thymidine assays described above. As a control for normal morphology,one well (well “A”) was left untreated (medium only). Incubation tookplace in a 37° C., 5% CO₂ humidified incubator. After each of the two (5minutes and 24 hours) exposure points, the therapeutic agent medium wasaspirated from each well, including the untreated well. One milliliterof fresh medium was then added to replace the aspirated medium.Re-incubation followed until each of the incremented evaluation pointswere achieved. At those points, the medium was aspirated andsubsequently replaced with 1 ml of 10% neutral buffered formalin for onehour to allow for proper fixation. These fixed slides were stained byhematoxylin (nuclear) and eosin (cytoplasmic) for morphologic evaluationand grading.

Results: The results of the 24 hour ³H-leucine protein inhibition assayand the 24 hour ³H-thymidine DNA synthesis inhibition assay are shown inFIGS. 10A-10D for suramin, staurosporin, nitroglycerin and cytochalasinB, respectively. All of the tested compounds showed an availabletherapeutic range (area under the curve of ³H-leucine assay is greaterthan that resulting from the ³H-thymidine assay), indicating usefulnessin the practice of sustained release dosage form embodiments of thepresent invention. More specifically, the compounds inhibited theability of vascular smooth muscle cells to undergo DNA synthesis in thepresence of 5% FBS to a greater extent than they inhibited proteinsynthesis of vascular smooth muscle cells. The protein and DNA synthesisinhibitory effects of suramin, staurosporin, nitroglycerin andcytochalasin B during a 5 minute and 24 hour pulsed exposure are shownin FIGS. 10A-D, respectively.

EXAMPLE 9 Specific Binding and Internalization of Targeted Particles byVascular Smooth Muscle Cells

The ability of vascular smooth muscle cells to bind and internalizeparticles coated with binding protein or peptide was demonstrated withmonoclonal antibody (NR-AN-01) coated gold beads both in vitro and invivo. The vascular smooth muscle cell tissue cultures (BO54), an antigenpositive control cell line (A375) and an antigen negative control cellline (HT29) were incubated with 10 nm gold beads, with one group coatedwith NR-AN-01 and a second, uncoated control group. The cells wereexposed to the beads as monolayer and cell suspension cultures, and wereexamined at six time points (i.e., 1 minute, 5 minutes, 15 minutes, 30minutes, 60 minutes and 24 hours) for binding and internalization byelectron microscopy.

Table 3 shows the results of the experimentation, indicating that thebinding to the cell surface is specific. The relative grading systemused throughout Table 3 represents a subjective assessment of particlebinding, wherein 0=none; 1=minimal; 2=mild; 3=moderate; and 4=marked. Ifaggregates of particles settled on the monolayer surface of both thesmooth muscle cells and the control cells, the particles werenonspecifically internalized by macro and micro phagocytosis. When thecells were maintained in a cell suspension, non-specific internalizationwas minimal or absent. Non-specific adherence of gold beads devoid ofNR-AN-01 to surface mucin produced by HT29 cells was observed, resultingin modest non-specific internalization thereof. Vascular smooth musclecell uptake of NR-AN-01 targeted gold beads was highly specific in cellsuspension cultures.

TABLE 3 Primary vesicle micro/macro Cell phagostasis secondaryendoplasmic Time Grid Product Cell Line Surface pinocytosis coated pitvesicle lysosome golgi reticulum Cell Monolayer 1 min Aa 05(G) A375 2 00 0 0 0 0 Ba 05(G) HT29 0 0 0 0 0 0 0 C 05(G) B054 2 1 0 0 0 0 0 Da (G)A375 0 0 0 0 0 0 0 Eb (G) HT29 0 0 0 0 0 0 0 F (G) B054 0 0 0 0 0 0 0 5min Ac 05(G) A375 4 1 0 0 0 0 0 Bb 05(G) HT29 0 0 0 0 0 0 0 Ca 05(G)B054 3 0 0 0 0 0 0 Dc (G) A375 0 0 0 0 0 0 0 Ea (G) HT29 0 0 0 0 0 0 0Fa (G) B054 0 0 0 0 0 0 0 15 min Aa 05(G) A375 3 1 0 0 0 0 0 Bb 05(G)HT29 0 0 0 0 0 0 0 Ca 05(G) B054 2 1 0 0 0 0 0 Da (G) A375 0 0 0 0 0 0 0Ea (G) HT29 0 0 0 0 0 0 0 Fa (G) B054 0 0 0 0 0 0 0 30 min A 05(G) A3754 3 0 2 0 0 0 B 05(G) HT29 0 0 0 0 0 0 0 C 05(G) B054 3 2 0 1 0 0 0 D(G) A375 0 0 0 0 0 0 0 E (G) HT29 0 1 0 0 0 0 0 F (G) B054 1 1 0 0 0 0 060 min Aa 05(G) A375 4 3 2 3 2 0 1 Ba 05(G) HT29 0 0 0 0 0 0 0 Cc 05(G)B054 3 2 0 2 0 0 1 Da (G) A375 0 1 0 0 0 0 1 Ec (G) HT29 1 1 0 1 0 0 0Fa (G) B054 1 2 0 1 0 0 0 24 hrs Ab 05(G) A375 2 1 1 2 4 0 2 Ba 05(G)HT29 0 1 1 2 3 0 0 Cc 05(G) B054 3 3 1 3 4 1 1 Da (G) A375 0 3 0 2 3 0 0Eb (G) HT29 0 3 0 3 1 0 0 Fb (G) B054 0 2 0 2 3 0 0 Cell Pellets 1 min1A 05(G) A375 2 0 0 0 0 0 0 7A 05(G) HT29 0 0 0 0 0 0 0 13A 05(G) B054 30 1 0 0 0 0 1B (G) A375 0 0 0 0 0 0 0 7B (G) HT29 0 0 0 0 0 0 0 13B (G)B054 0 0 0 0 0 0 0 5 min 2A 05(G) A375 3 1 0 0 0 0 0 8A 05(G) HT29 0 0 00 0 0 0 14A 05(G) B054 2 1 0 0 0 0 0 2B (G) A375 0 0 0 0 0 0 0 8B (G)HT29 0 0 0 0 0 0 0 15B (G) B054 0 0 0 0 0 0 0 15 min 3A 05(G) A375 4 1 01 0 0 0 9A 05(G) HT29 0 0 0 0 0 0 0 15A 05(G) B054 1 1 0 0 0 0 0 3B (G)A375 0 0 0 0 0 0 0 9B (G) HT29 0 0 0 0 0 0 0 15B (G) B054 0 0 0 0 0 0 030 min 4A 05(G) A375 4 2 0 0 0 0 0 10A 05(G) HT29 0 0 0 0 0 0 0 16A05(G) B054 2 1 0 0 0 0 0 4B (G) A375 0 0 0 0 0 0 0 10B (G) HT29 0 0 0 00 0 0 16G (G) B054 0 0 0 0 0 0 0 60 min 5A 05(G) A375 3 3 0 2 1 0 0 11A05(G) HT29 0 0 0 0 0 0 0 17A 05(G) B054 2 2 0 2 0 0 0 5B (G) A375 0 0 00 0 0 0 11B (G) HT29 0 0 0 0 0 0 0 17B (G) B054 0 0 0 0 0 0 0 24 hrs 6A05(G) A375 3 1 0 3 3 0 0 12A 05(G) HT29 0 0 0 0 0 0 0 18A 05(G) B054 2 10 1 3 0 0 6B (G) A375 0 0 0 0 0 0 0 12B (G) HT29 1 2 0 2 2 0 0 18B (G)B054 0 0 0 0 0 0 0

FIG. 11 shows a tangential section parallel to the inner surface of asmooth muscle cell characterized by numerous endocytic vesicles, severalof which contain antibody coated gold beads in the process of beinginternalized by the cell. These endocytic vesicles with particlesattached to cell surface antigens were stimulated to fuse with lysosomesat a higher than expected rate for normal cell surface membranerecycling. The resultant marked accumulation of internalized particleswas observed at the 24 hour time point and is shown in FIG. 12.

The targeted gold bead vascular smooth muscle cell surface binding,internalization and lysosome concentration was observed in vivo as well.NR-AN-01 coated gold beads were infused via intravascular catheter, openended with treated area occluded proximally and distally with slipligatures, at 3 atm pressure applied for 3 minutes into the wall of apig femoral artery immediately following balloon trauma. The beadinternalization rate varied with the degree of damage sustained by thevascular smooth muscle cell during the balloon trauma. Cells withminimal or no damage rapidly internalized the particles by endocytosisand phagocytosis, concentrating the internalized particles in lysosomes.Cells that were killed by the trauma exhibited surface bead binding.Cells that were damaged by the trauma but survived were characterized bybead surface binding with delayed internalization and lysosomeconcentration. FIG. 13 shows particulate concentration in the lysosomesin vivo at one week following bead administration.

EXAMPLE 10 Vascular Smooth Muscle in Vitro DNA and Protein SynthesisInhibition by Staurosporin and Cytochalasin

The ability of staurosporin and cytochalasin to inhibit in vitro DNA andprotein synthesis in vascular smooth muscle cells was tested. ³H-leucineand ³H-thymidine uptake and cytotoxicity assays were conducted inaccordance with the following protocols.

Cultured Cells:

BO54 cells (baboon smooth muscle cells) were derived from explants ofaortic baboon smooth muscle cells. Cells were expanded in DMEM(Dulbecco's Modified Eagle's Medium):F-12 medium (Whittaker Bioproducts,Walkersville, Md.) with 5% fetal bovine serum (FBS, Gibco) and 5% SerumPlus® (JRH Biologicals) (“complete medium”), and a seed lot of cells wasfrozen in liquid nitrogen for future use at passage seven.

5 Minute Exposure; Protein Synthesis Assay: Vascular smooth muscle cellsat 40,000-50,000 cells/ml were seeded and processed as described inExample 8, “5 minute exposure; ³H-leucine uptake.” Log dilutions ofstaurosporin (200 ng/ml, 20 ng/ml, 2 ng/ml, 0.2 ng/ml and 0.02 ng/ml)were dispersed in complete medium. For cytochalasin B, log dilutions at20 μg/ml, 2.0 μg/ml, 0.2 μg/ml, 0.02 μg/ml and 0.002 μg/ml weredispersed in complete medium. Complete medium was then added to thecontrol wells. One ml/well of each therapeutic agent dilution was addedin quadruplicate wells, and the agent of interest was incubated with thevascular smooth muscle cells for 5 min at room temperature in a sterileventilated hood. Following therapeutic agent incubation, the wells weresubsequently treated as described in Example 8, “5 minute exposure;³H-leucine uptake.”

5 Minute Exposure; DNA Synthesis Assay: Vascular smooth muscle (BO54)cells were seeded and processed in 24 well plates, as described aboveunder “5 Minute Exposure: Protein Synthesis Assay.” After 5 minincubation with the test therapeutic agent, the medium was aspirated and1 ml/well of 1.0 μCi/ml ³H-thymidine (rather than ³H-leucine) dispersedin complete medium was added. The cells were then incubated overnight at37° C. in a humidified, 5% CO₂ environment. The toxic effect of thetherapeutic agent was then determined, as described in the ProteinSynthesis Assay, above.

24 and 120 Hour Exposure; Protein Synthesis Assay: Vascular smoothmuscle (BO54) cells at 20,000 cells/ml were seeded in sterile 24 wellplates and incubated in complete medium (1 ml/well) overnight at 37° C.,5% CO₂, 95% air in a humidified atmosphere (saturation). Log dilutionsof staurosporin (100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0.01 ng/ml)were dispersed sequentially in the two media, as described below. Forcytochalasin B, log dilutions at 10 μg/ml, 1.0 μg/ml, 0.1 μg/ml, 0.01μg/ml and 0.001 μg/ml were dispersed sequentially in the two media, asdescribed below:

Medium (1)=Complete medium; and

Medium (2)=DMEM (leucine-free) with 0.5 μCi/ml ³H-leucine. Medium (2) isused for the final 24 hour incubation period of the experiment.

More specifically, in the 24 hour assay, each therapeutic agent wasdiluted in Medium (2), as noted above. Medium (1) was aspirated from thewells, and aliquots of therapeutic agent dilutions in Medium (2) wereadded in quadruplicate to the appropriate wells. Medium (2) was thenadded to the control wells.

In the 120 hour assay, each therapeutic agent was diluted in Medium (1),as noted above. Medium (1) was aspirated from the wells, and aliquots oftherapeutic agent dilutions in Medium (1) were added in quadruplicate tothe appropriate wells. Medium (1) was then added to the control wells.The medium was changed every 24 hours, and fresh therapeutic agent wasadded to the test wells. At 96 hr, (i.e., the fourth day), eachtherapeutic agent was diluted in Medium (2), as noted above. Medium (1)was aspirated from the wells, and aliquots of therapeutic agentdilutions in Medium (2) were added in quadruplicate to the appropriatewells. Medium (2) was then added to the control wells.

The test agents in ³H-leucine (and controls) were incubated overnight at37° C., 5% CO₂ in a humidified atmosphere. The toxic effect of thetherapeutic agents was then determined, as described in the 5 MinuteExposure: Protein Synthesis Assay, described above. In addition, thechanges in cells at each dilution were photographed using a Zeissmicroscope (Zeiss, West Germany) at 320×. The medium was then aspirated,and the cells were processed with TCA, as described above.

24 and 120 Hour Exposure; DNA Synthesis Assay: This assay was performedaccording to the procedure described for “24 and 120 Hour Exposure;Protein Synthesis Assay”, except Medium (2) in this 24 & 120 hr DNASynthesis Assay is:

Medium (2)=Complete Medium with 1.0 μCi/ml ³H-thymidine.

Medium (2) is used in the final 24 hour incubation of the experiment.

These protein and DNA synthesis assays are amenable for use with othertarget cell populations, especially adherent monolayer cell types.

Results: The minimum effective dose (MED) of each agent was determinedas a percentage of the control that was treated with medium only; 50% ofcontrol values was chosen as the cytotoxicity benchmark. At a 5 minexposure, staurosporin demonstrated an MED of 100 ng/ml in the proteinsynthesis assay and 1 ng/ml in the DNA assay. The 24 hour MED forstaurosporin was 10 ng/ml in the protein synthesis assay and 1 ng/ml inthe DNA synthesis assay. Both assays gave an MED of 1 ng/ml for a 120hour exposure of staurosporin.

At a 5 minute exposure, cytochalasin B demonstrated an MED of 10 μg/mlin the protein synthesis assay as well as in the DNA assay. The 24 hourMED for cytochalasin B was 1.0 μg/ml in the protein synthesis assay and0.1 μg/ml in the DNA synthesis assay. Both assays gave an MED ofapproximately 0.1 μg/ml for a 120 hour exposure of staurosporin.

Cytochalasin C and cytochalasin D therapeutic agents were tested at 24and 48 hour exposures using the same dilutions as described forcytochalasin B, above. At 24 hours, cytochalasin C demonstrated an MEDof 1.0 μg/ml in the protein synthesis assay and an MED of 0.01 μg/ml inthe DNA synthesis assay. At 48 hours, cytochalasin C demonstrated an MEDof 0.1 μg/ml in the protein synthesis assay and 0.01 μg/ml in the DNAsynthesis assay. Cytochalasin D demonstrated an MED of 1.0 μg/ml in the24 hour protein synthesis assay and an MED of 0.1 μg/ml in the 24 hr DNAsynthesis assay. A 48 hour exposure to cytochalasin D gave an MEDranging between 0.1 and 0.01 μg/ml in both the protein synthesis and DNAsynthesis assays.

EXAMPLE 11 Vascular Smooth Muscle Cell Migration Inhibition

Scratch assays to determine the extent of smooth muscle cell migrationinhibition by cytochalasin B were performed in accordance with thefollowing protocol:

Vascular smooth muscle cells (BO54) were derived from explants of baboonaortic smooth muscle, as described in Example 10. The cells were grownin flat bottom, six well tissue culture plates, which hold about 5 ml ofmedium. The vascular smooth muscle cells were plated at 200,000cells/well and placed at 37° C. in a humidified 5% CO₂ incubator for 18hours. The wells were then scratched with a sterile portion of a singleedge razor blade that was held by clamp or pliers and was broughtaseptically into contact with the bottom of the well at a 90° angle. Thecells from a small area along the scratch were removed by a sterilecotton tipped applicator while the blade was in contact with the bottomof the well. After incubation, the presence of cells in the “scratched”area is indicative of cell migration across the scratch line. A controlincubation showed significant cellular migration, and serves as thestandard against which the migration of cells exposed to the therapeuticagent is compared.

Briefly, a stock solution of cytochalasin B (Sigma Chemical Co.) indimethyl sulfoxide (DMSO) at 1 mg/ml was prepared. Test dilutions ofcytochalasin B or control medium were added. Each experiment includedtwo sets of plates:

A set: Test agent exposure for 1, 3, 6, 8 and 10 days only; and

B set: Test agent exposure for 1, 3, 6, 8 and 10 days, followed by aseven day recovery time with control medium.

Both sets of plates were fixed (10% formalin in PBS) and stained (0.02%crystal violet) at the end of the timed exposures. Test concentrationsfor cytochalasin B were 1, 0.1 and 0.01 μg/ml, and a negative mediumcontrol was included. Fresh medium and drug were supplied 3 times perweek.

Table 4 shows the results of these experiments. In this Table, “M”indicates Migration Grade, wherein −=no migration; +1=minimal; +2=mild;+3=moderate; and +4=marked (maximum density; limit of cell contactinhibition) migration of vascular smooth muscle cells into the clearedarea adjacent to the scratch. In this Table, “T” denotes a morphologicalToxicity Grade, wherein −=no toxicity; +1=minimal; +2=mild; +3=moderate;and +4=marked toxicity. The migration results are expressed as “Grade inthe Cleared Area of the Well/Grade in an Undisturbed Region of theWell.” The toxicity values represent a grade for all cells in each well.

The data indicate that cytochalasin B inhibits the migration (+1 to +2)of vascular smooth muscle cells into the cleared area adjacent to thescratch at a dose of 0.1 μg/ml with only minimal (− to +1) morphologicaltoxicity. The data also show that the treated cells (0.1 μg/ml) regainthe ability to migrate (+3 to +4) following removal of the therapeuticagent, even after 10 days of continuous exposure to the therapeuticagent.

TABLE 4 SCRATCH-MIGRATION ASSAY: INHIBITION OF VASCULAR SMOOTH MUSCLECELL MIGRATION BY CYTOCHALASIN B Continuous Exposure 7-day Recovery PostExposure Dosage μg/mL Dosage μg/mL Day Control 0.0 0.01 0.1 1.0 Control0.0 0.01 0.1 1.0 1 M +1/+3 +1/+3 +1/+3 —/+2 +3/+4 +3/+4 +3/+4 +2/+3 T —— — +3 — — — +2 3 M +3/+4 +3/+4 +1/+4 —/+2 +3/+4 +3/+4 +3/+4 +2/+3 T — —+1 +3 — — — +1 6 M +3/+4 +3/+4 +2/+4 —/+2 +4/+4 +4/+4 +3/+4 +2/+3 T — —+1 +4 — — — +3 8 M +3/+4 +3/+4 +2/+4 —/+2 +4/+4 +4/+4 +3/+4 +2/+3 T — —+1 +4 — — — +3 10 M +3/+4 +3/+4 +2/+4 —/+2 +4/+4 +4/+4 +4/+4 +2/+3 T — —+1 +4 — — — +3

EXAMPLE 12 Therapeutic Agent Cytotoxic Effects on Vascular Smooth MuscleCells—Pulse and Continuous Exposure

Vascular smooth muscle cells were exposed to a therapeutic agent in oneof two exposure formats:

Pulse exposure: The pulse exposure protocol is described in Example 8above (see “Morphological Cytotoxicity Evaluation—Pulsed Exposure”).

Continuous exposure: The same methodology is used for continuousexposure morphological cytotoxicity evaluation as for the pulseexposure, except that the test wells were continuously exposed totherapeutic agent in medium during the exposure period. The medium andtherapeutic agent were aspirated from each well daily, including fromthe untreated control well, and were replaced with 1 ml of fresh mediumand therapeutic agent (or medium alone for control wells). Re-incubationfollowed, until each of the incremental evaluation points of the longterm continuous exposure protocol was achieved. These incrementalevaluation time points were at 6, 24, 48, 72, 96, 120, 168, 216 and 264hours. At the designated time period, the appropriate cells were fixed,stained and evaluated as in the pulse exposure protocol. The results ofa continuous exposure experiment are shown in Table 5 for suramin,staurosporin and cytochalasin B. The 5 min and 24 hr data presented inTable 5 are correlates of the data contained in FIGS. 10A, 10B and 10C.

TABLE 5 MORPHOLOGICAL CYTOTOXICITY ASSAY Drug & Dose Cytochalasin BSuramin Staurosporine Exposure Protocol 10 μg 1 μg 0.1 μg 0.01 μg 10 mg1 mg 0.1 mg 0.01 mg 100 ng 10 ng 1 ng 0.1 ng 5 min + 2 hrs 0.5 0 0 — 0 00 — 0 0 0 — 5 min + 6 hrs 4 1 0 — 1 0 0 — 0 0 0 — 5 min + 24 hrs 4 0.5 0— 1 0 0 — 0 0 0 — 5 min + 48 hrs 4 1 0 — 2 0 0 — 2 1 0 — 5 min + 72 hrs4.5 1 0 — 3 1 0 — 3 1.5 0 — 5 min + 96 hrs 5 1 0 — 3 1 0 — 3.5 1.5 0 — 5min + 120 hrs 5 1 0 — 3 1 0 — 4 1.5 0 — Continuous 6 hrs — 3 0 0 3 1 0 —0 0 0 0 Continuous 24 hrs — 3 1 0 3 2 0 — — 0 0 0 24 hrs + 24 hrs — 30.5 0 4 3 0 — — 0.5 0 0 24 hrs + 48 hrs — 4 1 0 4 3 0 — — 2 0 0 24 hrs +72 hrs — 4 0.5 0 4 3 0.5 — — 1 0 0 24 hrs + 96 hrs — 4 0 0 4 3.5 1 — —1.5 0 0 24 hrs + 120 hrs — 4 0 0 — — — — — 1.5 0 0 Continuous 24 hrs — 30 0 — 1 1 0 — 3 1 0 Continuous 48 hrs — 3 1 0 — 3 2 0 — 3 2 0 Continuous72 hrs — 3 1 0 — 4 3 0 — 3 2 0 Continuous 96 hrs — 3 2 0 — 4 3 0 — 3 2 1Continuous 120 hrs — 3 1 0 — 5 4 0 — 3 2 1 Continuous 168 hrs — 4 1 0 —5 4 0 — 3 2 1 Continuous 216 hrs — 4 1 0 — 5 4 0 — 3 2 1 Continuous 264hrs — 4 1 0 — 5 4 0 — 4 2 1

At an in vitro effective dosage, cytochalasin B (1 μg/ml; ananti-migration/contraction effective dose) and staurosporin (1 ng/ml; ananti-proliferative effective dose) exhibited a cytotoxicity grade of 1(minimal) and 2 (mild), respectively. Independent studies have indicatedthat a grade of 3 (moderate) or less is preferred for a cytostatic,anti-proliferative agent of the present invention.

EXAMPLE 13 In Vivo BRDU Assay: Inhibition of Vascular Smooth Muscle CellProliferation

BRDU assay: In vivo vascular smooth muscle proliferation was quantitatedby measuring incorporation of the base analog 5-bromo-2′-deoxyuridine(BRDU, available from Sigma Chemical Co.) into DNA during cellular DNAsynthesis and proliferation. BRDU incorporation was demonstratedhistochemically using commercially available anti-BRDU monoclonalantibodies. The 1 hour pulse labeling permits assessment of the numberof cells undergoing division during the pulse period.

The BRDU pulse labeling protocol described above is used as a standardevaluation technique with in vivo pig vascular studies. Followingsurgical and treatment procedures (discussed, for example, in Examples 7and 11 herein) and a post-surgical recovery period, pigs were sedatedand pulsed with BRDU 1 hour prior to tissue collection.

Briefly, the pigs were sedated with tiletamine hydrochloride andxylazine (as in Example 7, “Gross Pathology and HistologicalEvaluation”) by intramuscular injection. BRDU was then administeredintravenously via the lateral ear vein. Two ml of BRDU at aconcentration of 50 mg/ml was administered to each 30-40 lb pig. Onehour later, the pigs were sacrificed by intravenously administeredpentobarbital. Test artery segments were then removed (a segmentincluded normal vessel located proximally and, if possible, distallywith respect to the treated artery segment). The artery segments weretransected at 2 mm intervals; arranged in order in cryomolds with O.C.T.(optimum cutting temperature) compound (Tissue Tek®, Miles Laboratories,Inc., Elkhart, Ind.); and frozen in liquid nitrogen. The blocks weresectioned at 5 microns and immunohistologically stained to detect BRDUusing the following procedure.

BRDU-labeled cell detection: After BRDU (1 g BRDU diluted in 17 mlsterile water and 3 ml 1 N NaOH) pulse labeling and test artery segmentremoval and sectioning (as above), immunohistochemical staining withanti-BRDU monoclonal antibody provides a visual means of determining amitotic index over a specified time period. The immunohistochemicalstaining method was performed as follows:

1) 5 μm sections of test artery were dehydrated in cold acetone (−20°C.) for 10 minutes;

2) Sections were mounted on glass microscope slides, and the slides weredried in a 37° C. oven for 10 minutes;

3) Slides were rehydrated in PBS for 10 minutes;

4) Slides were subjected to Feulgen's acid hydrolysis using 1 N HCl,wherein two aliquots of 1 N HCl are preheated to 37° C. and 60° C. priorto proceeding;

5) Slides were rinsed with 1 ml of 1 N HCl at 37° C. for 1 min;

6) Slides were transferred to 60° C. 1 N HCL for 15 min;

7) Slides were rinsed with 1 ml of 1 N HCl at 37° C. for 1 min;

8) Slides were washed with room temperature PBS, using 3 changes of PBSat 5 min intervals;

9) Endogenous, cross-reactive sites on the sections were blocked withnormal goat serum (1:25 in PBS) for 20 min;

10) Slides were washed with PBS, as in step 8;

11) Sections were incubated with mouse anti-BRDU antibody (DAKOCorporation, Carpinteria, Calif.) at 10 μg/ml for 30 min;

12) Slides were washed with PBS, as in step 8;

13) Sections were incubated with horseradish peroxidase-labeled (HRPO)goat anti-mouse IgG₁ (Jackson Immunoresearch Laboratories, Inc., WestGrove, Pa.; diluted 1:20 in PBS) and 4% human AB serum for 30 min;

14) Slides were washed with PBS, as in step 8;

15) Sections were incubated with chromogen (3,3′-diaminobenzidine (DAB;Sigma) at 5 mg/ml in 200 ml PBS) and 200 μl of 30% H₂O₂ for 10 min;

16) Slides were washed with PBS, as in step 8;

17) Samples were counterstained with Gill I hematoxylin (Gill I LernerLaboratories, Pittsburgh, Pa.; 30 dips);

18) Slides were washed with PBS, as in step 8; rinsed with a bluingsolution (1 gm lithium carbonate in 500 ml dH₂O); washed with deionizedwater; and

19) Test samples were then dehydrated, cleared and coverslipped.

At the conclusion of this procedure, a positive immunohistological stainexhibits a brown color at the site(s) of reactivity.

Cytocidal agents inhibited BRDU uptake relative to a PBS control;however, cytochalasin B and staurosporin inhibited BRDU uptake (i.e.,cell proliferation) without killing the vascular smooth muscle cells.The number of vascular smooth muscle cells labeled with BRDU wasassigned a grade at 400× magnification as follows:

1=≦1/high power field (HPF);

2=2 to 5/HPF;

3=>5 to ≦10/HPF; and

4=>10/HPF.

Both cytochalasin B and staurosporin inhibited proliferation for 24hours following balloon trauma (grade 1), yielding a BRDU labeling gradeequivalent to that of a pre-trauma baseline (grade 1). PBS andmonoclonal antibody controls exhibited grade 2.5 to 4 BRDU labelingduring the same time period. At 4 days post-trauma, arteries treatedwith cytochalasin B or staurosporin, as well as PBS and monoclonalantibody controls, exhibited a BRDU labeling grade of 4. Theanti-proliferative, non-cytocidal properties of cytochalasin B andstaurosporin suggest that these agents are amenable to sustained releasedosage formulations for reduction of vascular stenosis.

EXAMPLE 14 Direct Conjugation of NR-AN-01 Antibody to CarboxylicFunctional Groups of a Latex Particle

Antibody-coated latex particles (a model of an antibody-coated,sustained release dosage form) may be obtained using the followingaseptic technique:

Conjugation:

To 4 ml 0.05 M sodium borate, pH 8.5, containing 0.01% Tween-20®(polyoxyethylene sorbitan monolaurate, Sigma) is added 0.5 ml PBScontaining 5 mg NR-AN-01 monoclonal antibody. To this solution at roomtemperature is added, with vortexing, 2.5 ml of an aqueous suspensioncontaining 50 mg of 1 μm diameter carboxylated latex particles.Immediately thereafter, 0.50 ml of water containing 100 mg of freshlydissolved 1(3-dimethyl-aminopropyl)3-ethyl carbodiimide HCl is addedwith vortexing. The solution is then incubated with shaking for 1-2 hrat room temperature. The reaction mixture is then diluted with 50 ml of50 mM phosphate buffer, pH 6.6, containing 0.2% gelatin stabilizer(phosphate/gelatin buffer). The mixture is centrifuged at 40,000×g for 2hr at 4-10° C. The supernatant is decanted, and the pellet isresuspended in 50 ml phosphate/gelatin buffer using low level sonicationfor 10 sec. Centrifugation is repeated, and the pellet is resuspendedtwo times, followed by resuspension in the phosphate/gelatin buffer. Theconjugated particles are then lyophilized using standard protocols andsorbitol excipients.

Characterization:

(a) Sizing: Particle size homogeneity is assessed by laser anisotropyor, for particles larger than 1 μm, by microscopic examination.

(b) Specific Binding Assessment: Specific binding to smooth muscle cellsis determined by histological examination of tissue or cell pelletmicrotome slices after incubation of protein/peptide conjugates withconjugated particles, with or without blocker protein/peptide includedin the incubation mixture. Preferred detection techniques include secondantibody assays (i.e., anti-mouse Ig) or competition assays (i.e.,radioscintigraphic detection in conjunction with radioisotopicallylabeled protein/peptide conjugates).

(c) Assessment of the extent of protein/peptide derivitization: Thisdetermination is performed by coating the latex particles withradioisotopically labeled antibody, followed by detection ofradioactivity associated with the coated particles.

The characterization of antibody-coated particles is described in Table6.

TABLE 6 Characterization of NR-AN-01-Coated Latex Particles ParticleOffering of μg Ab Bound/ Ab Molecules Diameter Ab/particle 5 mg LatexPer Particle 1.2 μm 40,000  42 3520 1.2 μm 84,000  66 5470 0.4 μm 32,000 99 3160 0.4 μm 64,000 140 4550 0.1 μm   932 140  65

The particle aggregation effect of pH during antibody conjugation ispresented in Table 7.

TABLE 7 Effect of pH During Antibody Conjugation - Particle AggregationParticle pH* During Particle Aggregation** Diameter Conjugation +Tween20 ® −Tween 20 ® 1.2 μm 8.5 <5% <2.5% 1.2 μm 7.0 ≈20% ≈10% 1.2 μm 5.510% 100% 0.4 μm 8.5 <10% <5% 0.4 μm 7.0 ≈30% ≈20% 0.4 μm 5.5 100% 100%0.1 μm 8.5 <20% <10% 0.1 μm 7.0 ≈50% ≈40% 0.1 μm 5.5 100% 100% *Using 50mM MES (pH 5.5); phosphate (pH 7.0); or borate (pH 8.5) buffer, asdescribed. **As assessed by microscopic examination, on a scale of0-100%.

These data suggest that proteins or peptides may be directly conjugatedwith sustained release dosage forms of the present invention. Morespecifically, poly-lactic/glycolic acid particulates having terminalcarboxylic acid groups will be conjugated according to the proceduredescribed herein or the alternative procedures described in thespecification hereof.

EXAMPLE 15 In Vivo Studies of Cytochalasin B

Biodistribution of Cytochalasin B. To determine the biodistribution ofcytochalasin B, mice were injected (i.p.) with 50 mg/kg cytochalasin B.Control mice were injected with DMSO/Tween 20/carboxymethyl cellulose(“vehicle”). The mice were sacrificed at 3, 12, 24 and 72 hours aftercytochalasin B or vehicle administration. Organs were removed,homogenized, extracted and the amount of cytochalasin B in tissuesquantitated by HPLC. About 75% of the cytochalasin B remained in theperitoneal cavity or at the injection site. Of the organs tested, thehighest amount of cytochalasin B was found in the liver. Subsequentanalyses showed that the maximum tolerated dose for cytochalsin B was 50mg/kg, and that this dose may be administered every second day.

Cytochalasin B was also administered intravenously to mice at 3.5 mg/kg(in methanol or Tween 20/carboxymethyl cellulose). Mice were sacrificedat 2 minutes, 15 minutes, 30 minutes, 3 hours and 12 hours aftercytochalasin B administration and tissue extracts analyzed forcytochalasin by TLC. The maximal recovery of cytochalasin B from tissueextracts was 32%. The data showed that cytochalasin B was localized tothe lung and the injection site, and that 3.5 mg/kg of cytochalasin Bresulted in no acute toxicity. By 12 hours after administration, therewere very low levels of cytochalasin B in tissues.

³H-cytochalasin B (2 μg; 30 μCi/μg) was injected i.v. into BALB/c micehaving urinary bladders that had been externally ligated. Animals weresacrificed at 15 minutes, 30 minutes, 2 hours or 16 hourspost-injection. Organs were removed, blotted, weighed, air dried andassayed for radioactivity. Fifty—73% of the total injected dose wasaccounted for in the tissues sampled (blood, heart, brain, muscle, bone,lung, liver, spleen, stomach, kidney, intestines, and urinary bladder).Clearance of ³H-cytochalasin B from the blood was extremely rapid withless than 1% of the injected dose in circulation by 15 minutes. Onlyliver, skeletal muscle and intestines showed significant retention of ³Hactivity. All tissues had clearance of ³H activity to below 1.5%injected dose per gram of tissue by 16 hours.

Cytochalasin B Metabolism. ³H-cytochalasin B at a dose of 1.5 or 8 μg/mlwas mixed with viable or non-viable human liver slices and media, andthe amount of ³H-cytochalasin B in media or tissue assessed by HPLC(Tables 8 and 9).

The cytotoxity of ³H-cytochalasin B and its subsequent metabolites wasalso assessed by evaluating dose dependent changes in mitochondrialfunction by monitoring3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT)activity following a 24 hour exposure of human liver slices to the testagent (Table 10).

TABLE 8 DISTRIBUTION OF ³H CYTOCHALASIN B MEDIA AND HUMAN LIVER TISSUE³H Cyto- Incuba- Incubation Period % Activity % Activity chalasin B tionat 37° C. in in (μg/ml) System (Hours) Media Tissue 1.5 Media/viable 184 16 1.5 tissue 4 84 16 1.5 24  91  9 8 Media/viable 1 88 12 8 tissue 484 16 8 24  89 11 8 Media/boiled 1 83 17 8 tissue 4 87 13 8 24  87 13

TABLE 9 HUMAN LIVER SLICES METABOLISM STUDY NATURE OF TRITIUM ACTIVITYEXTENT OF METABOLIC CONVERSION % Meta- ³H Cyto- Incubation % Non Ex- %Ex- bolized of chalasin B Incubation Period tractable tractableExtractable (μg/ml) System (Hours) Activity Activity Activity 8 Media/ 10.5 99.5 24.2 8 viable 4 1 99.0 59.4 8 tissue 24  1.3 98.7 98.0 1.5Media/ 1 0.5 99.5 43.0 1.5 viable 4 0.6 99.4 77.0 1.5 tissue 24  0.499.6 98.7 8 Media/ 1 0.5 99.5 5 8 boiled 4 1 99.0 5 8 tissue 24  1.398.7 5 8 Media 1 NA NA 5 8 Only 4 NA NA 4 8 24  NA NA 5

TABLE 10 MTT Absorbance in Human Liver Slices Exposed to ³H cytochalasinB for 24 hours. Dose Level (μg/ml) MTT Absorbance 0   0.639 +/− 0.188(control) 0.1 0.844 +/− 0.014 1.5 0.841 +/− 0.081 8.0 0.850 +/− 0.082^(a)MTT absorbance values reflect the mitochondrial viability in whichhigh absorbance values represent viable mitochondria and while lowabsorbance values reflect nonfunctional mitochondria. Each valuerepresents the mean +/− standard deviation optical density of triplicateliver samples.

The results in Tables 8-9 indicated that 98% of the ³H-cytochalasin Bwas metabolized within 24 hours of administration with greater than 80%of the total reactivity being present in the media and less than 20% inthe tissue. The results in Table 10 indicated that ³H-cytochalasin B orits subsequent metabolites was not cytotoxic.

To determine the metabolism of ³H-cytochalasin B in human blood,³H-cytochalasin B (8 μg/ml) was dissolved in saline; 1:1 dilution ofsaline and human plasma; or 1:1 dilution of saline and human wholeblood. The mixtures were incubated for 20 hours at 37° C. and thenanalyzed by HPLC for stability and metabolism. ³H-cytochalasin B was notmetabolized when mixed with either saline or plasma. However,³H-cytochalasin B was metabolized by human whole blood with themetabolite having an HPLC retention time and profile consistent withthat seen in the human liver slice assay (see above).

Toxicity Studies. To determine the toxicity of cytochalasin B, rats (4male and 4 female) were injected with 10 μg/ml cytochalasin B four timesa day for 7 days (Table 11). Data regarding body weight changes, foodconsumption, food efficiency, hematology parameters, coagulationparameters, serum chemistry parameters, and gross necropsy findings werecollected. The only parameter which suggested an adverse effect ofcytochalasin B administration was an elevation of the mean relativeheart weight in treated female animals. There were no gross ormicroscopic changes detected in the heart to account for the elevation.Thus, daily administration of cytochalasin B to rats at a dose of 800μg/kg (for a total injected dose of 5600 μg/kg) may have an effect onheart weight of female (but not male) rats. However, clotted blood wasnot routinely removed from the heart lumens prior to weighing the heart.

TABLE 11 Group Number of Animals Dose Level Dosing Dosing Number MalesFemales Treatment (μg/mL) Route Frequency Duration 1 4 4 Biostent  0 IV4 times/day 7 days Control (2 hrs apart) 2 4 4 Cytochalasin B 10 IV 4times/day 7 days (2 hrs apart)

To determine the toxicity of chronic cytochalasin B administration,cytochalasin B was administered intravenously to Sprauge-Dawley rats forseven days (Table 12). Data regarding food consumption, body weights,hematology parameters, clinical chemistry parameters, coagulationprofiles, organ weights, clinical observations, gross necropsy findingsand histopathology were collected. It was found that chronic intravenousadministration of cytochalasin B at doses up to 600 μg/kg/day did notresult in any indication of adverse effects or toxicity.

TABLE 12 Dose Group Number of Animals Level Dosing Dosing Number MalesFemales Treatment (μg/mL) Route Frequency Duration  1* 15 15 Control 0Intravenous 4 times/day 7 days 2 10 10 Cytochalasin B 1.3 injection (≈2hrs apart) 7 days 3 10 10 3.0 7 days  4* 15 15 7.5 7 days  5* 15 15 7.57 days *Five animals/sex/group were used for a 14-day nontreatmentrecovery period and were euthanized on day 22 **Five animals/sex wereeuthanized on days 8, 15, and 22

A similar study in dogs (Table 13), which collected data on foodconsumption, body weights, hematology parameters, clinical chemistryparameters, coagulation profiles, organ weights, clinical observations,thoracic cavity ausculation, opthalmic examination, urinalysis, grossnecropsy findings, and histopathology (high dose group), found thatintravenous administration of cytochalasin B for seven consecutive daysto beagle dogs at doses up to 648 μg/kg/day (a cumulative dose of 4,536μg/kg) did not result in any indication of adverse effects or toxicity.

TABLE 13 Dose Volume Number of Dose (mL/kg); Group Animals Level FlowRate Dosing Dosing Number ♂ ♀ Treatment (μg/mL) (mL/min) Route FrequencyDuration  1* 6 6 Control 0 20; 10 IV 4 times/day 7 days (=6 hrs apart) 24 4 Cytochalasin B 1.3 20; 10 IV 4 times/day 7 days (=6 hrs apart) 3 4 4Cytochalasin B 4.4 20; 10 IV 4 times/day 7 days (=6 hrs apart)  4* 6 6Cytochalasin B 8.1 20; 10 IV 4 times/day 7 days (=6 hrs apart)  5** 6 6Cytochalasin B 8.1 20; 10 IV 4 times/day 7 days (=6 hrs apart) ♂ =males; ♀ = females; IV = intravenous infusion *Two dogs/sex/group wereheld for a 14-day nontreatment recovery period and were euthanized onday 23 **Three dogs/sex were euthanized on days 9 and 23.

EXAMPLE 16 Biological Stenting of Balloon Traumatized Pig Arteries

Pig femoral arteries were traumatized as described in Example 7, andthen treated with cytochalasin B. About 1.5 to about 2 ml ofcytochalasin B at 0.1 μg/ml was infused into portions of the artery thathad been separated from other portions by ligatures. The artery was thenpressurized for 3 minutes and the fluid aspirated. Approximately 8 toabout 30 lambda of the solution is retained in the interstitial spacesurrounding the cells in the tunica media. Ten femoral arteries (twoarteries obtained from each of the 5 pigs that were treated according tothe single dose protocol described in Example 7) were then evaluatedhistologically at 4 days or 3 weeks after cytochalasin B adminstration.The maximal luminal area of each artery was measured and calculated fromdigitized microscopic images by a BQ System IV computerized morphometricanalysis system (R & M Biometrics, Inc., Nashville, Tenn.). Thisexperiment was repeated with 5 additional pigs (two arteries per pig;cytochalasin B dose=0.1 μg/ml, applied for 3 minutes at 1 atm pressure;same time points). The data obtained from the two experiments werecombined. Balloon traumatized pig arteries that had been treated withcytochalasin B displayed a larger luminal area at the 4 day and 3 weekpost-treatment time points, as compared to arteries treated with othertest agents or controls.

The luminal area of the traumatized and cytochalasin B-treated segmentsof the arteries were also compared to the luminal area of the normal,untreated region of the femoral artery proximal to the test area. Theresults showed that the lumen area in the test region was approximatelytwo times as large as the area of the normal control segment of the sameartery. The negative control agents, PBS and monoclonal antibodyNR-AN-01, showed no increase or a slight decrease in lumen area ascompared to the normal control segment of the same artery.

A cytochalasin B dose response study was then conducted on 10 pigs,following the experimental protocol described in Example 7. Briefly,both femoral arteries in each of 2 pigs were treated with one of thefollowing doses of cytochalasin B: 0.0 μg/ml (i.e., PBS negativecontrol); 0.01 μg/ml; 0.10 μg/ml; 1.0 μg/ml; and 10.0 μg/ml as describedabove. The agent was delivered by intraluminal catheter at 1 atmpressure for 3 minutes, and the arteries were evaluated 3 weeks later bythe morphometric analysis system described above. The ratio of treatedartery luminal area to proximal normal artery luminal area wasdetermined as a percent change in treated vs. normal area, or the arterylumen size (diameter or cross sectional area) of treated arteriesrelative to to traumatized but untreated arteries. A significantthreshold effect was observed at doses from 0.1 μg/ml (about a 140%increase) to 1.0 μg/ml (FIG. 14). The 10 μg/ml dose appeared to be toxicto the vascular smooth muscle cells. The subthreshold dose (0.01 μg/ml)and negative control (PBS) exhibited about a ±20% change in luminalarea. However, subsequent experiments (see below) demonstrated that both0.01 μg/ml and 10 μg/ml doses were efficacious.

Electron micrographs revealed that within one hour of balloon trauma,there was a depolymerization of the myofilaments in traumatized vessels,which are contractive organelles. The depolymerization of themyofilaments is a normal physiological response of vascular smoothmuscle cells to trauma, and is the first step in their transformationfrom a contractile to a secretory and migratory cell. Treatment oftraumatized swine arteries with about 0.1 to about 10.0 μg/ml ofcytochalasin B did not result in an increased rate, or more extensivedepolymerization, of the myofibrils. However, electron micrographsshowed that the myofilament reformation was retarded. Based on thesustained increase in vessel diameter, the return to normal vascularcontractility may be slowed more extensively than is suggested by theretarded return to normal morphology.

The traumatized and treated areas of the artery did not undergo theconstriction or chronic geometrical (vascular) remodeling that normallyoccurred in sham controls in the pig, and which has been described inman, following PTCA. Cross sections of the artery showed a largercross-sectional area of the total vessel, obtained by measuring theratio of the vessel area inside the external elastic lamina (EEL) of thetreated area to the mean of the total vessel area of the proximal anddistal regions of the same artery, compared to arteries traumatized butnot treated with cytochalasin B. In arteries that were traumatized witha torquable balloon that damages the vessel wall without tearing thetunica media, there was a more uniform intimal proliferation and thelarger vessel size (area inside the EEL) resulted in a larger luminalcross-sectional area and a significant decrease in restenosis. When thevessels were extensively damaged and the tunica media was torn into,there was extensive proliferation and thrombus remodeling that resultedin highly variable intimal proliferation.

Thus, the administration of cytochalasin B to swine vascular smoothmuscle cells by infusion catheter following balloon dilation traumaresulted in a more extensive retention of the artery lumen size(diameter or cross-sectional area) than was produced by the dilatingballoon. This effect was achieved by replacing the entire interstitialfluid volume between cells of the tunica media with the therapeuticagent.

Moreover, cytochalasin B has a wide therapeutic index which ranges fromabout 0.1 to 10 μg/ml, with no evidence of toxicity at 10 μg/ml. Tenμg/ml is the maximum saturation concentration of cytochalasin B insaline. Furthermore, the effect produced by cytochalasin Badministration became more apparent over the 3 to 8 weeks following theballoon trauma. These data suggest that cytochalasin B acts as a“biological stent” when delivered to traumatized arteries.

Balloon traumatized swine femoral arteries from control and treated (0.1μg/ml cytochalasin B) were evaluated at 1, 4, 7, 14, or 21 days afterintervention. Morphometric analysis on frozen histologic sections ofartery showed that cytochalasin B treated arteries reached a state ofsustained dilation which changed very little between days 7 and 21. Atwo way analysis of variance indicated a statistically significantdifference (p<0.05) in the artery lumen areas over three weeks betweenthe cytochalasin B treated and diluent control groups.

A dose-response study of balloon traumatized swine coronary arteriesshowed that treatment with cytochalasin B at 0.1, 1.5 or 10.0 μg/ml(Table 14, “Biostent”) resulted in sustained arterial dilation of thecoronary luminal area at three weeks after intervention. Moreover,cytochalasin B administration did not result in myocardial or arteriallesions attributable to the cytochalasin B as evaluated histologically.No statistically and biologically significant changes attributable tocytochalasin B were seen in clinical chemistry parameters, hematologyparemeters, body weights, blood pressure or electrocardiograms.

TABLE 14 SWINE CORONARY MORPHOMETRY DATA P-value Group Days Mean % StdUnpaired Dose (μg/ml)* Post Surgery Lumen Area Dev T-test Saline  4 96.515.2 — 0.1 μg/mL  4 97.0 17.1 0.48  Biostent 1.5 μg/mL  4 101.7 6.80.28  Biostent  10 μg/mL  4 103.9 14.1 0.25  Biostent Saline 21 78.719.1 — 0.1 μg/mL 21 98.0 15.5 0.004 Biostent 1.5 μg/mL 21 100.9 18.50.002 Biostent  10 μg/mL 21 110.9 19.2 0.004 Biostent *Dose is based onthe concentration of cytochalasin B (μg/ml) in the about 8 to about 30lambda volume of fluid delivered to about 10 to about 20% of the tunicamedia.

Swine coronary arteries were also traumatized by embolectomy orover-sized PTCA balloon, and then 8-16 ml of cytochalasin B at 8.0 μg/mlwas infused into the arterial wall with a MIC catheter to achieve atherapeutic dose. Controls included a diluent control and a traumatizeduntreated control, and all animals were sacrificed 4 weeks afterintervention and coronary arteries were fixed by perfusion. Morphometrywas performed on selected sections from proximal, treated and distalsegments of coronary arteries (Table 15).

TABLE 5 Swine Coronary Artery Study Neointimal Luminal Arterial AreaArea Area Group Area std Area std Area std Untreated 1.88 1.62 0.77 0.382.19 1.07 Control Saline Treated 1.39 1.13 0.73 0.37 1.88 0.96 Control8.0 μg/ml 1.56 1.24 0.58 0.30 1.80 0.70 Cytochalasin B

While the data shown in Table 15, in contrast to previous studies,showed that the local delivery of cytochalasin B did not result in astatistically significant increase in luminal area, the data does showthat there was a trend toward beneficial arterial remodeling asevidenced by larger arterial area bounded by the external elastic laminain the cytochalasin B treated arteries when compared to either of thecontrols. The diameter of, or the area within, the EEL of the artery canbe compared to controls as an indicator of the degree of vascularremodeling. The lack of a statistically significant increase in luminalarea may be due to increased sample variablility, increased neointimalformation, and/or increased degree of trauma.

In summary, these studies demonstrate that the administration of acytoskeletal inhibitor, such as cytochalasin B, in an amount which canbiologically stent a traumatized vessel may also be efficacious toinhibit or reduce the proliferation of vascular smooth muscle cells.

EXAMPLE 17 Sustained Release Formulations of Cytochalasin B and Taxol

To determine the efficacy of the local, sustained release dosage formsof cytochalasin B or taxol to inhibit restenosis, cytochalasin B ortaxol in a supporting structure, e.g., a “wrap,” was applied to theadventitial tissue surrounding a balloon traumatized rabbit carotidartery (Groups 1-11) or a balloon traumatized pig femoral artery (Group12) (Table 16).

The arteries in the animals in Group 1a and 1b were treated with 20 mgof cytochalasin B in 1 g of a bovine collagen gel (BioCore, Inc.,Topeka, Kans.) that was supported by, or enclosed in, a bovine collagenmesh wrap (Skin Temp-Biosynthetic Skin Dressing, BioCore, Inc., Topeka,Kans.). At 1 week post treatment, the cytochalasin B treated artery inanimal 1233 showed no intimal or adventital proliferation. There wasmarked cell death in the outer zone of the tunica media with heterophilsinfiltrating the tunica media. Heterophils were present outside thewrap, but cytochalasin B inhibited heterophils and macrophages frominfiltrating the wrap. The artery of the control (1249) animal hadmoderate intimal proliferation, and heterophils and histiocytes wereinfiltrating into the wrap. Cell death in the tunica media was minimal.

At 2 weeks post-treatment, there was minimal intimal and adventitalproliferation in the cytochalasin B wrap-treated area of the artery(animal 1224). The intima was loosely arranged and there was minimalheterophil infiltration. Syncytial giant cells were present. In theartery of the control wrap animal (animal 1222), there was moderateintimal proliferation with heterophils and macrophage in the wrap area.These cells were visible in the tunica media.

At three weeks post-treatment, there was no intimal proliferationobserved in the cytochalasin B wrap-treated area of the artery (1244).Heterophils and syncytial giant cells were present around the wrap.There was significant necrosis of the cells in the tunica media withinfiltrating heterophils and macrophages. No endothelium was present. Inthe control (animal 1232) artery, there was marked intimalproliferation, with well organized adventitia and perivascular tissue.Heterophils and macrophages were infiltrating the wrap. The cells in thetunica media were viable and there was a mural thrombus in the vessellumen. Thus, inhibition of intimal proliferation was seen in thearteries of Group 1a and 1b animals treated with cytochalasin B,however, there was significant reaction to the wrap material.

The arteries in the animals in Group 2a and 2b were treated withcytochalasin B (30% wt/wt; 300 mg cytochalasin B/g silicone) in asilicone wrap (Q-7 4840, Dow Coming, Midland, Mich.). One weekpost-trauma there was no significant intimal or adventitialproliferation of smooth muscle cells (SMCs) or mesenchymal tissue(animal 1229). There was significant necrosis of the SMCs in the outerzone of the tunica media. In areas that appeared to have been minimallytraumatized by the torquable ballon there was minimal to no cellularnecrosis. This indicated that traumatized cells were more prone to diewhen exposed to this dose of cytochalasin B but that this dose was notcytocidal to minimally traumatized or normal SMCs. There was minimalmononuclear and polymorphoneuclear cell infiltration into the tunicamedia. A few heterophils were seen infiltrating from the vessel lumen.

In the control animal (1228), there was less cellular necrosis in thetunica media, and the necrosis present was located in the inner zonerather than the outer zone of the artery wall. Thus, cytochalasin Binhibition of cellular repair appears to increase tunica media necrosis.The control also lacked tunica media or adventitial proliferation andorganization of the perivascular clot. Cellular infiltration in any areawas minimal.

Two weeks after initiating cytochalasin B treatment there was completeinhibition of intimal proliferation and only minimal perivascular clotorganization which was primarily due to fibrin formation and notmesenchymal proliferation (animal 1227). There was mild infiltration ofpolymorphonuclear cells and minimal infiltration of mononuclear cellsinto the tunica media and adventitia. No endothelium was present in thewrap area, except for a few small isolated foci. The control artery(animal 1226) had moderate intimal proliferation and adventitialproliferation with mesenchymal organization of the perivascular clotarea. Foci of endothelial proliferation were larger and more extensivein the control animal compared to the cytochalasin B treated vessel.

With 3 weeks exposure (animal 1212) to cytochalasin B in a siliconewrap, the vessel showed marked cell loss in the tunica media which wasmost severe in the outer zone. Cellular infiltration in the tunica mediaand adventitia was minimal and endothelilzation was only present in afew focal areas. There was moderate, irregular intimal proliferation;however, the intimal cells and what few endothelial cells that werepresent were unorganized and lack polarity. The inhibition of migrationby cytochalasin B resulted in this loss of organization or polarity. Theintimal proliferation was also mild in the control vessel (1230);however, the intima was well organized and was almost completelyendothelized. There was mininal cell loss from the tunica media. Thus,significant intimal inhibition was seen in the first two weeks in Group2 treated animals.

The vessels in the animals in Group 3a and 3b were treated with 8 mgcytochalasin B in 100 mg of a pluronic gel (F-127, BASF) that wassupported by a 1 cm×1 cm bovine collagen mesh wrap. One week aftertreatment, the cytochalasin B treated artery of animal 1250 had mildintimal proliferation that was irregular in thickness. There wasapproximately 30% re-endothelization and the tunica media cells wereviable with the most significant loss (mild) being in the inner zone ofthe tunica media. There was a marked pyogranulomatous reaction to thepluronic gel in the perivascular and adventitial region. Completethrombosis of the control artery from animal 1261 prevented itsevaluation.

At two weeks, the pluronic gel with cytochalasin B stimulated a markedpyogranulomatous reaction in the adventitial and perivascular tissue.There was mild, irregular intimal proliferation and completeendothelization. The tunica media cells were viable. There appeared tobe a mild cell loss from the inner zone of the tunica media. The arteryof the control animal (1247) had mild, irregular intimal proliferation,complete endothelization with plump endothelial cells and viable cellsin the tunica media. There was marked pyogranulomatous inflammatoryreaction to reminants of the pluronic gel.

At three weeks, the arteries of animals 1248 and 1246 showed reminantsof the collagen wrap; however, the wrap was less resolved in thecytochalasin B treated animal (1248) than in the control (1246). Thisretardation of wrap resorbsion may result from cytochalasin B inhibitionof macrophage migration and function. Both the treated and control had amoderate amount of intimal hyperplasia at 3 weeks, so there was nosignificant amount of intimal inhibition by cytochalasin B whenadministered in the pluronic gel. Foci of dystrophic mineralization wereseen in the cytochalasin B treated artery. Thus, no inhibitory effect onthe intima was observed in these animals (Group 3) at 1, 2 or 3 weeksafter the initiation of treatment.

The arteries of the animals in Group 4a and b that were treated with 100mg cytochalasin B (10% wt/wt) in a 1 g silicone wrap had significantinhibition of intimal proliferation at all time points. In thecytochalasin B wrap-treated artery of animal 1259, there was no intimalproliferation or adventitial fibrosis present at one week aftertreatment. Ectatic vessels were present in the adventitia and theperivascular clot was unorganized and composed of only fibrin. There wasmarked cell loss from the tunica media, especially in the outer zone.Heterophils were seen infiltrating the tunica media. In the controlartery (1206), there was no intimal proliferation at 1 week; however,there was early fibrous organization of the perivascular clot. Cellularloss from the tunical media was more diffuse than in the cytochalasin Bwrap which was most severe in the outer zone.

The cytochalasin B wrap-treated artery of animal 1253 had minimalintimal proliferation at two weeks, compared to the control (1258)wrap-treated artery which had maximal intimal proliferation. The intimalproliferation in the cytochalasin B wrap-treated artery was irregularand appeared to be the result of organizing mural thombi by infiltratingSMC. There was only loose thin layers of platelets in the cytochalasin Bwrap-treated artery, with margination of heterophils. There was noendothelization in the cytochalasin B wrap-treated artery and <20%endothelization in the control artery. The perivascular clot wasunorganized and remained fibinous in the cytochalasin B wrap-treatedartery and well organized in the control artery. The control artery hadminimal cell loss from the tunica media while the cytochalasin Bwrap-treated artery had marked cell loss.

At three weeks, the cytochalasin B wrap-treated artery (1251) showedminimal to no intimal proliferation. The intimal proliferation appearedto occur where there was less cell loss from the tunica media. There wasearly re-endothelization in the cytochalasin B wrap-treated artery, butthe cells were often rounded, loosely attached and only a few scatteredfoci were present (<10%). The perivascular clot in the cytochalasin Bwrap-treated artery was unorganized and still consisted of fibrin,whereas the control artery was well organized with fibroblasts andcollagen matrix. The control artery was completely thrombosed, there wasmarked initmal production in distal areas of the thrombus which wereless completely organized.

The arteries in the animals in Group 5a and b were treated with 50 mgtaxol in 1 g of a silicone wrap (5% wt/wt). This treatment showed markedinhibition of intimal proliferation at all time points. The taxolwrap-treated artery (animal 1278 at 1 week) had no intimal oradventitial proliferation and the perivascular clot was fibrinous andunorganized. There was a marked loss of tunica media cells and noendothelial lining present. The control (1279) had mild intimalproliferation with very early fibrosis of the fibrinous perivascularclot. The lumen was approximately 85% re-endothelized. Both the treatedand control arteries had mild heterophil infiltration into the tunicamedia and adventitia.

The artery from animal 1281, which had been treated for at two weekswith taxol, had no intimal proliferation, minimal adventitial fribrosisand marked cell necrosis in the tunica media with mild heterophilinfiltration. Focal areas of necrosis and dystrophic mineraliztion werepresent in the adventitia and perivascular clot tissue. The artery fromthe control animal (1280) had moderate intimal proliferation, withmarked organization of the adventitia and perivascular clot. The lumenwas 100% re-endothelized and the tunica media SMCs were viable in thecontrol artery.

At three weeks, the taxol wrap-treated artery (1242) had no intimalproliferation and was 50% re-endothelized with plump appearingendothelial cells. There was minimal organization of the perivascularclot and marked cell loss from the tunica media. There was mildinfiltration of heterophils into the tunica media and marginating on thevessel lumenal surface. The control artery (1234) had marked intimalproliferation and fibrosis of the adventitia and perivascular clot.Cells in the tunica were viable.

In Group 6a and 6b, taxol-treated arteries also had a marked inhibition2 weeks after the wrap was removed. Animal 1276 had a taxol wrap for 2weeks, then the wrap was removed and the animal sacrificed 3 weekslater. Following the 3 week recovery period from the taxol wrap removal(1276) there was only minimal intimal proliferation, except in a fewfocal areas that appeared to be thickened due to SMC organization ofmural thrombi, in this artery. The adventitia was well organized andthere was a significant cell loss in the tunica media but the cellspresent were viable. The lumen was approximately 90% re-endothelized.The control (1277) artery had marked intimal proliferation, wellorganized perivascular and adventical tissue and was 100%re-endothelized.

The results observed for Group 7a animals demonstrated that cytochalasinB-treated arteries (10% wt/wt) showed no intimal proliferation for 2weeks. The decrease in the release rate of the cytochalasin B, however,resulted in a mild intimal proliferation by week three after wrapplacement. At one week (1257), the arteries showed no intimalproliferation, and a marked necrosis of tunical media SMCs with moderateheterophil infiltration. There was no endothelium and heterophils andmacrophages were marginated along the lumen surface. Moreover, there wasno evidence of platelet aggregates adhering to vessel wall. At two weeks(1265), the arteries were similar morphologically to the one weekarteries. By three weeks (1266), the arteries showed mild irregularintimal proliferation. Furthermore, heterophils were rare in the tunicamedia, the lumen was 70% re-endothelized and there was early fibrosis inthe adventitia with unorganized perivascular clot still present, in thetreated arteries. This indicated that by 3 weeks the level oftherapeutic agent had fallen below therapeutic level within the arterywall; however, there was still enough drug to have an inhibitory effecton clot organization immediately adjacant to the wrap.

The arteries of Group 8a animals, which were treated with 10%cytochalasin B for 2 weeks, then the wrap was removed and vesselsevaluated 2 weeks later, had variable intimal proliferation within andbetween animals. The artery of animal 1254 had variable intimalproliferation which ranged from none to mild, well developed adventitialfibrosis, marked cell loss in the tunic media and focal areas ofdystrophic mineralization in the outer tunica media and adventitia. Themild intimal proliferation areas were at the ends of the wrap area,suggesting an infiltration from the adjacent untreated artery regions.The lumen was approximately 60% re-endothelized. The artery of animal1255 had mild to moderate intimal proliferation, viable cells in thetunica media and well organized tissue in the adventitia. The lumen was100% re-endothelized. The artery in animal 1256 was completelythrombosed. Proximal to the chronic thrombus in the area of the wrap wasan acute thrombus and there was moderate intimal proliferation.

While there was moderate intimal proliferation in the arteries of someanimals, the proliferation in these arteries was still less than thecontrols in Group 9b. The mannitol control silicone wraps were on theartery for two weeks following balloon trauma and then removed and theanimal necropsied and the artery histologically evaluated 1 weekfollowing wrap removal. Two of the arteries (1267 and 1268) had moderateintimal proliferation with 100% re-endothelization and one had maximumproliferation. The one with maximum intimal prolifertion had an acuteoccluding thrombus present.

The arteries in the animals in Groups 10 and 11 were treated with 10 mgcytochalasin B loaded in 1 g of a silicone wrap (1% wt/wt) that wasapplied to the artery for 2 weeks, surgically removed, andhistologically evaluated 2 or 4 weeks later, respectively. Nosignificant difference was seen by qualitative evaluation between thetest and control animals. Animals 1304 and 1305 had a cytochalasin B(1%) wrap for 2 weeks which was then removed. Two weeks after theremoval the animal was sacrificed. The artery from animal 1304 showedmoderate initmal proliferation in most areas of the wrap, in areas ofmarked tunica media cell necrosis and wall dystrophic mineralization theproliferation was mild. There was 100% re-endothelization and noheterophils were present in the intima or tunica media. The adventitiaand perivascular clot area was well organized. The artery from animal1305 was similar to the artery from animal 1304 morphologically. Theartery from animal 1306 showed marked intimal proliferation, noinfiltrating heterophils in the intima or tunica media and was 100%re-endothelized.

Animals 1307, 1308 and 1309 were exposed to a cytochalasin B (1%) wrapfor 2 weeks which was then removed. Four weeks after removal the animalswere sacrificed. The artery from animal 1307 had moderate initmalproliferation with focal areas of thickening due to mural thrombusorganization by SMCs. There was significant loss of cells from thetunica media and the elastic elamina appear collapsed. A few heterophilswere present in the adventitia. There were areas or sections in the wraparea with minimal intimal proliferation. The artery from animal 1308showed moderate intimal proliferation with areas of marked cell loss inthe tunica media and dystrophic minearalization in the outer zone of thetunica media. The vessel was 100% re-endothelized. The artery fromanimal 1309 had marked intimal proliferation with a well organizedaventiticia and perivascular region. Animal 1311 was not evaluated dueto thrombosis. The results of the artery from animal 1312 were quitevariable, with sections showing a range of intimal proliferation, frommild to moderate. Endothelization appeared to be complete in thesearteries.

The arteries from Group 12 animals (pig femoral arteries) that weretreated with 30% wt/wt cytochalasin B loaded silicone wraps showedsignificantly intimal proliferation for the first two weeks. While therewas intimal proliferation in the arteries 3 weeks later, theproliferation was still less than the proliferation observed for thecontrols.

TABLE 16 SURGERY NECROPSY TIME POST ANIMAL # GROUP # TREATMENT DATE DATETREATMENT GRADE 1233  1a CytoB + Bovine col gel + col mat 2/28/96 3/7/961 wk 1249  1b Control + Bovine col gel + col mat 2/28/96 3/7/96 1 wk 31224  1a CytoB + Bovine col gel + col mat 2/28/96 3/15/96 2 wks 2 1222 1b Control + Bovine col gel + col mat 2/28/96 3/15/96 2 wks 3 1244  1aCytoB + Bovine col gel + col mat 2/28/96 3/20/96 3 wks 1 1232  1bControl + Bovine col gel + col mat 2/28/96 3/20/96 3 wks 3 1229  2aCytoB 30% + silicone 2/22/96 2/28/96 1 wk 0 1228  2b Control + silicone2/22/96 2/28/96 1 wk 2 1227  2a CytoB 30% + silicone 2/22/96 3/7/96 2wks 0 1226  2b Control + silicone 2/22/96 3/7/96 2 wks 3 1212  2a CytoB30% + silicone 2/22/96 3/15/96 3 wks 2 1230  2b Control + silicone2/22/96 3/15/96 3 wks 2 1250  3a CytoB + pluronic gel +col. wrap 3/7/963/15/96 1 wk 2 1261  3b Control + pluronic gel + col. wrap 3/7/963/15/96 1 wk NA 1245  3a CytoB + ploronic gel + col. wrap 3/7/96 3/20/962 wks 2 1247  3b Control + pluronic gel + col. wrap 3/7/96 3/20/96 2 wks2 1248  3a CytoB + pluronic gel + col. wrap 3/7/96 3/28/96 3 wks 3 1246 3b Control + pluronic gel + col. wrap 3/7/96 3/28/96 3 wks 3 1259  4aCytoB 10% − mannitol + silicone 4/8/96 4/15/96 1 wk 0 1260  4b Control −mannitol + silicone 4/8/96 4/15/96 1 wk 0 1253  4a Cytott 10% −mannitol + silicone 4/8/96 4/22/96 2 wks 1 1258  4b Control − mannitol +silicone 4/8/96 4/22/96 2 wks 4 1251  4a CytoB 10% − mannitol + silicone4/8/96 4/29/96 3 wks 0 1252  4b Control − mannitol + silicone 4/8/964/29/96 3 wks 4 1278  5a Taxol + silicone 3/20/96 3/2//96 1 wk 0 1279 5b Control − silicone 3/20/96 3/28/96 1 wk 2 1281  5a Taxol + silicone3/20/96 4/4/96 2 wks 1280  5b Control − silicone 3/20/96 4/4/96 2 wks 31242  5a Taxol + silicone 3/7/96 3/28/96 3 wks 0 1243  5b Control −silicone 3/7/96 3/28/96 3 wks 4 1276  6a Taxol + silicone 3/20/964/23/96 2 wks-3 wks 1 1277  6b Control − silicone 3/20/96 4/23/96 2wks-3 wks 4 1257  7a Cytob 10% − mannitol = silicone 5/16/96 5/23/96 1wk 0 1265  7a Cytob 10% − mannitol = silicone 5/16/96 5/29/96 2 wks 01266  7a Cytob 10% − mannitol = silicone 5/16/96 6/5/96 3 wks 2 Note:see control cases 1260 = 1 wk 1258 = 1252 = 3 wks 2 wks 1254  8a CytoB10% − mannitol + silicone 5/16/96 6/11/96 2 wks—2 wks 0-2 1255  8a CytoB10% − mannitol + silicone 5/16/96 6/11/96 2 wks—2 wks 2-3 1256  8a CytoB10% mannitol + silicone 5/16/96 6/11/96 2 wks—2 wks 3 1267  9b Control −mannitol + silicone 5/23/96 6/14/96 2 wks-1 wks 3 1268  9b Control −mannitol + silicone 5/23/96 6/14/96 2 wks-1 wks 3 1269  9b Control −mannitol + silicone 5/23/96 6/14/96 2 wks-1 wks 4 1304 10a CytoB 1% −mannitol + silicone 6/10/96 7/9/96 2 wks—2 wks 3 1305 10a CytoB 1% −mannitol + silicone 6/10/96 7/9/96 2 wks—2 wks 3 1306 10a CytoB 1% −mannitol + silicone 6/10/96 7/9/96 2 wks—2 wks 4 1307 11a CytoB 1% −mannitol + silicone 6/10/96 7/22/96 2 wks-4 wks 3 1308 11a CytoB 1% −mannitol + silicone 6/10/96 7/22/96 2 wks-4 wks 3 1309 11a CytoB 1% −mannitol + silicone 6/10/96 7/22/96 2 wks-4 wks NA 1310 11b Control −mannitol + silicone 6/10/96 7/22/96 2 wks-4 wks 4 1311 11b Control −mannitol + silicone 6/10/96 7/22/96 2 wks-4 wks NA 1312 11b Control −mannitol + silicone 6/10/96 7/22/96 2 wks-4 wks 3 1029LF 12c CytoB 30% −silicone wrap 2/1/96 2/22/96 3 wks 2 1020RF 12f Control silicone wrap2/1/96 2/22/96 3 wks. 3 1030LF 12e CytoB 30% − silicone wrap 2/7/962/22/96 2 wks. 1 1030RF 12d Control silicone wrap 2/7/96 2/22/96 2 wks.4 1036LF 12a CytoB 30% − silicone wrap 2/1/96 2/7/96 1 wk. 1 1036RF 12bControl silicone wrap 2/1/96 2/7/96 1 wk. 1

In summary, intimal proliferation of traumatized pig arteries wassignificantly inhibited with both cytochalasin B and taxol in sustainedrelease dosage form. The best controlled sustained release oftherapeutic agent, without the stimulation of secondary inflammatoryreaction, was obtained with an adventitial wrap material comprisingsilicone. The silicone wraps inhibited intimal proliferation with 30%and 10% loadings of cytochalasin B; however, as the level of releasedrops off between 2 and 3 weeks there was initiation of intimalproliferation. When wraps were left in place for 2 weeks then surgicallyremoved and the arteries examined from 1 to 4 weeks later, thereappeared to be an intimal proliferation rebound effect. The reboundeffect occurred when the intimal proliferation of the artery treatedwith the therapeutic agent approaches, but is still less than, theintimal proliferation in the control artery. The animal treated withtaxol appeared to have less of a rebound effect than the cytochalasin Btreated arteries.

EXAMPLE 18 Delivery of Crystalline Cytochalasin B or Taxol

The in vivo tissue distribution of cytochalasin B administered incrystalline form was evaluated in balloon traumatized swine femoralarteries after local delivery. A femoral artery of a Yorkshire crossbredswine was balloon traumatized by overinflation and rotation of aVascu-Flo™ Silicone embolectomy catheter. Balloon trauma was immediatelyfollowed by intravascular delivery of 10 μg/ml ³H-cytochalasin Bcrystals (Sigma Chemical Co., St. Louis, Mo.) in saline (saturated) forthree minutes under 1 atm of pressure. Blood flow was resumed in theartery for five minutes prior to sacrifice of the animal. An analysis ofthe tissue distribution of ³H-cytochalasin B showed that this method waseffective at delivering 31 ug of ³H-cytochalasin B which localizedpredominantly to the adventitia. ³H-cytochalasin B was visualizedhistologically by the presence of silver grains in an autoradiographicemulsion. Thus, these results showed that crystalline cytochalasin B canbe delivered locally to a vessel wall in vivo.

Another study employed twenty, male, Sprague-Dawley rats. The ratsunderwent balloon trauma to their left carotid artery, followed byinter-arterial infusion of a solution containing 1 mg crystallinecytochalasin B in 300 ml vehicle (Hanks sterile salt solution with 0.5%Cremophor) or a diluent (saline) control. Animals were sacrificedimmediately after infusion, and 2, 4, 7 and 14 days post-trauma andinfusion. Post-sacrifice, the left and the right (control) carotidarteries were removed. Samples of arteries were obtained forquantitation of ³H-cytochalasin B by oxidation and scintillationcounting, histopathology, autoradiography and vascular morphometry.Histopathology documented uniform, circumferential balloon trauma in thearterial wall of the left carotid arteries.

Autoradiographically, cytochalasin B crystals were present on day 0 inintraluminal fibrin clots, adherent to the intima but rarely present inthe adventitia. By day 2, the number of crystals diminished compared today 0, and by day 4 crystals were not detectable by autoradiography. Theautoradiographic results correlated closely with quantitative assessmentof ³H-cytochalasin B by oxidation and scintillation counts in whichapproximately 8 ug of cytochalasin B was present over the treated lengthof artery on day 0 and slightly less than 2 ng was present by day 2.However, one of the two animals sacrificed on day 4 still hadcytochalasin B levels above background. Morphometric analysis of leftcarotid arteries of crystalline cytochalasin B treated rats compared todiluent treated rats showed no statistically significant reduction inneointima formation. However, the five treated rats had a higher meanluminal area and a smaller neointimal area than diluent treated control.

Cytochalasin B and taxol were administered periadventially. Three groupsof seven adult male rats underwent balloon trauma of the left carotidartery immediately followed by periadventitial placement of eithercytochalasin B crystals (7.8-11.8 mg/rat), taxol crystals (3.4-6.5mg/rat), or no drug (control). The cytochalasin B and taxol crystalswere placed in a uniform pattern which covered the surgically exposedsurface of the carotid artery, followed by closure of surroundingsubcutaneous skin and tissues by sutures. Fourteen days later rats weresacrificed and their carotid arteries processed for histologic andmorphometric analysis.

Two cytochalasin B treated animals died due to acute hemorrhage at thesurgical site and hypovolemic shock prior to the 14 day sacrifice point.Two additional cytochalasin B treated and one taxol treated animal weresacrificed with rapidly enlarging subcutaneous swelling and hemorrhageat the surgical site prior to the 14 day sacrifice point. All animalstreated with either cytochalasin B or taxol crystals had significanttoxicity at the surgical site which was characterized by varying degreesof hemorrhage, necrosis of the vessel wall, necrosis of adjacentskeletal muscle and inflammation. In addition, both the taxol andcytochalasin B treated animals had a delay in post-surgical weight gain.

The three cytochalasin B treated, 6 taxol treated and 7 control animalswhich survived to the 14 day sacrifice point were evaluatedmorphometrically. Taxol treated animals had statistically significantlylarger luminal areas and no neointimal proliferation when compared tothe balloon traumatized, untreated control animals in a two-tailedt-test with p<0.05. Cytochalasin B treated animals showed no statisticaldifference from the controls in luminal area, neointimal area, medialarea, areas bounded by the internal and external elastic lamina orintimal to medial ratio.

To further evaluate the efficacy of crystalline taxol to inhibitneointimal formation in rats, four groups of 5-6 adult male ratsunderwent balloon trauma of the left carotid artery followed immediatelyby periadventitial delivery of 1, 0.1, 0.01 or 0 mg of taxol crystals in500 mg of a pluronic polymer in gel matrix. Fourteen days later, therats were sacrificed, serum was collected and their carotid arterieswere processed for histologic and morphometric analysis.

Five animals (3—1 mg and 2—0.01 mg) died post-surgically due totechnical difficulties. Grossly, myonecrosis of the adjacent skeletalmuscle (pale white regions of the musculature) was present in 3/3, 1/5,0/4 and 0/5 animals in the 1 mg, 0.1 mg, 0.01 mg and control groups,respectively. Histologically, myonecrosis was confirmed in the adjacentskeletal muscle and in some regions of the tunica media of the leftcarotid artery in the 1 mg treatment group but not in the other groups.Morphometrically, there was no statistical significance in luminal area,neointimal area, area bounded by the internal elastic lamina, area ofthe tunica media, area bounded by the external elastic lamina orneointimal/medial ratio when compared by analysis of variance using theexcell data analysis software package.

Periadventitial treatment of rat carotid arteries with 1 mg taxolcrystals in 500 mg of a pluronic gel resulted in gross myonecrosis ofthe adjacent musculature. While the number of animals surviving in thisgroup was too low to assess for statistical significance in thereduction of neointimal formation, neointimal area was 38% less thanthat of control animals.

For animals treated with 0.1 and 0.01 mg taxol, a reduction in theirneointimal area and neointimal/medial ratio was observed when comparedto control animals, although this did not reach statistical significancegiven the small number of animals per group. Moreover, animals in thelower dose groups showed no (0.01 mg), minimal (0.1 mg) or limited (1.0mg) toxicity, indicating that lower doses may be efficacious and exhibitfewer adverse side effects than doses greater than 1.0 mg.

All publications and patents are incorporated by reference herein, asthough individually incorporated by reference, as long as they are notinconsistent with the disclosure. The invention is not limited to theexact details shown and described, for it should be understood that manyvariations and modifications may be made while remaining within thespirit and scope of the invention defined by the claims.

What is claimed is:
 1. A method for inhibiting or reducing diminution invessel lumen area of a mammalian blood vessel comprising introducing tothe vessel a therapeutic shunt comprising an amount of a cytoskeletalinhibitor effective to inhibit or reduce stenosis or restenosis of thevessel following placement of the shunt.
 2. The method of claim 1wherein the cytoskeletal inhibitor comprises taxol, a cytochalasin, oran analog thereof.
 3. The method of claim 1 wherein the cytoskeletalinhibitor comprises taxol.
 4. The method of claim 1 wherein thecytoskeletal inhibitor comprises an analog of taxol.
 5. The method ofclaim 1 wherein the amount inhibits vascular smooth muscle cellproliferation.
 6. The method of claim 1 wherein the cytoskeletalinhibitor is releasably embedded in, coated on, or embedded in andcoated on, the shunt.
 7. The method of claim 1 wherein the cytoskeletalinhibitor is releasably embedded in, coated on, or embedded in andcoated on, a non-liquid matrix.
 8. The method of claim 7 wherein thematrix is a gel, paste or permeable membrane.
 9. The method of claim 1wherein the cytoskeletal inhibitor is coated on the shunt.
 10. Themethod of claim 1 wherein the cytoskeletal inhibitor is releasablyembedded in microparticles or nanoparticles, or a mixture thereof. 11.The method of claim 1 wherein the cytoskeletal inhibitor is insubstantially crystalline form.
 12. A method for inhibiting or reducingdiminution in vessel lumen area of a mammalian blood vessel comprisingintroducing to the vessel a stent overlayed with an artificial graftwhich artificial graft comprises an amount of a cytoskeletal inhibitoreffective to inhibit or reduce stenosis or restenosis of the vesselfollowing placement of the stent.
 13. The method of claim 12 wherein thecytoskeletal inhibitor comprises taxol, a cytochalasin, or an analogthereof.
 14. The method of claim 12 wherein the cytoskeletal inhibitorcomprises taxol.
 15. The method of claim 12 wherein the cytoskeletalinhibitor comprises an analog of taxol.
 16. The method of claim 12wherein the amount inhibits vascular smooth muscle cell proliferation.17. The method of claim 12 wherein the cytoskeletal inhibitor isreleasably embedded in, coated on, or embedded in and coated on, thegraft.
 18. The method of claim 12 wherein the cytoskeletal inhibitor isreleasably embedded in, coated on, or embedded in and coated on, anon-liquid matrix.
 19. The method of claim 18 wherein the matrix is agel, paste or permeable membrane.
 20. The method of claim 12 wherein thecytoskeletal inhibitor is coated on the graft.
 21. The method of claim12 wherein the cytoskeletal inhibitor is releasably embedded inmicroparticles or nanoparticles, or a mixture thereof.
 22. The method ofclaim 12 wherein the cytoskeletal inhibitor is in substantiallycrystalline form.
 23. A method for inhibiting or reducing diminution invessel lumen area of a mammalian blood vessel comprising introducing tothe vessel a therapeutic adventitial wrap comprising an amount of acytoskeletal inhibitor effective to inhibit or reduce stenosis orrestenosis of the vessel following placement of the wrap.
 24. The methodof claim 23 wherein the cytoskeletal inhibitor comprises taxol, acytochalasin, or an analog thereof.
 25. The method of claim 23 whereinthe cytoskeletal inhibitor comprises taxol.
 26. The method of claim 23wherein the cytoskeletal inhibitor comprises an analog of taxol.
 27. Themethod of claim 23 wherein the amount inhibits vascular smooth musclecell proliferation.
 28. The method of claim 23 wherein the cytoskeletalinhibitor is releasably embedded in, coated on, or embedded in andcoated on, the wrap.
 29. The method of claim 23 wherein the cytoskeletalinhibitor is releasably embedded in, coated on, or embedded in andcoated on, a non-liquid matrix.
 30. The method of claim 29 wherein thematrix is a gel, paste or permeable membrane.
 31. The method of claim 23wherein the cytoskeletal inhibitor is releasably embedded inmicroparticles or nanoparticles, or a mixture thereof.
 32. The method ofclaim 23 wherein the cytoskeletal inhibitor is in substantiallycrystalline form.
 33. A method for inhibiting or reducing diminution invessel lumen area of a mammalian blood vessel comprising introducing tothe vessel a therapeutic artificial graft comprising an amount of acytochalasin effective to inhibit or reduce stenosis or restenosis ofthe vessel following placement of the graft.
 34. The method of claim 33wherein the cytochalasin comprises cytochalasin A.
 35. The method ofclaim 33 wherein the cytochalasin comprises cytochalasin B.
 36. Themethod of claim 33 wherein the cytochalasin comprises an analog ofcytochalasin B.
 37. The method of claim 33 wherein the cytochalasincomprises cytochalasin D.
 38. The method of claim 33 wherein thecytochalasin is releasably embedded in, coated on, or embedded in andcoated on, the graft.
 39. The method of claim 33 wherein thecytochalasin is releasably embedded in, coated on, or embedded in andcoated on, a non-liquid matrix.
 40. The method of claim 33 wherein thecytochalasin is in substantially crystalline form.